The Wellcome Trust Centre for Cell Biology2012

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The Wellcome Trust Centre for Cell Biology

2012

The expansion of research in cell biology was planned in 1992 as a result of the vision of Professor Sir Kenneth Murray, who was at the time Biogen Professor at the Institute of Cell and Molecular Biology. A seed contribution of £2.5 million from the Darwin Trust was followed by financial commitments from The Wolfson Foundation, the University and the Wellcome Trust, allowing construction of the Michael Swann Building. The majority of research space was earmarked for Wellcome Trust-funded research. Recruitment, based on research excellence at all levels in the area of cell biology, began in earnest in 1993, mostly but not exclusively, through the award of Research Fellowships from the Wellcome Trust. The Swann Building was first occupied by new arrivals in January 1996 and became “The Wellcome Trust Centre for Cell Biology” from October 2001.

Historical Background

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Director’s Introduction 4

Robin Allshire 6

A. Jeyaprakash Arulanandam 8

Jean Beggs 10

Adrian Bird 12

Atlanta Cook 14

William C. Earnshaw 16

Kevin G. Hardwick 18

Lea A. Harrington 20

Adele Marston 22

Gracjan Michlewski 24

Hiro Ohkura 26

Juri Rappsilber 28

Kenneth E. Sawin 30

Eric Schirmer 32

Irina Stancheva 34

David Tollervey 36

Mike Tyers 38

Malcolm Walkinshaw 40

Julie Welburn 42

Steven West 44

Outreach and Public Engagement 46

List of Groups 48

Centre Publications 2010 - 2012 53

International Scientific Advisory Board 60

Content

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The Wellcome Trust Centre for Cell Biology (WTCCB) is one of nine UK-based Wellcome Trust funded Centres, three of which are in Scotland. The Centre was granted this status by the Wellcome Trust in 2001, the year that the draft human genome was published. The 18 research groups comprising the WTCCB occupy the Michael Swann Building on the Kings Building Campus of the University of Edinburgh. The Swann Building was constructed in the mid 1990s as a purpose built centre for research in molecular cell biology. From its inception until October 2011, the WTCCB was directed by Prof. Adrian Bird. During this time, Adrian’s outstanding work had established the scientific excellence and international reputation of the Centre.

I have taken on the responsibility of directing the WTCCB in an exciting period, when major changes are underway in the field of cell biology. One of the great triumphs of nineteenth century biology was the “Universal Cell Theory” - the understanding that all living creatures - plants, animals and humans - are formed from minute structures called cells. The basic premise of the discipline of cell biology is that living cells are more than extremely small test tubes. The many biochemical reactions taking in place in all cells form pathways that are highly organised; physically, in space and in time. The human body is, astonishingly, made up of some 50 trillion cells, every one of which contains an entire copy of the human genome and all the machinery needed to duplicate itself. Although small, human cells are immensely complex. Understanding how cells function normally is key to understanding the defects that arise during infection and disease – and remains a key challenge for 21st century biology.

Results from cell biology tell us that almost all genes and proteins function within highly complex cellular pathways. Repair of defects caused by infection or disease therefore needs system-level intervention. This, of course, requires understanding of entire cellular pathways and the wider systems within which they operate. In response to these challenges a global scientific endeavour is underway that is changing the way that Cell Biology is studied. The WTCCB is among the leading international centres of research in this field. Researchers in the WTCCB are attempting to understand and integrate events from molecular interactions all the way up to systems-level behaviour. The core of our work is hypothesis-driven research, with groups applying a wide range of different techniques to the problems analysed; these include microscopy, genetics,

Director’s Introduction

Professor David Tollervey, Director Wellcome Centre for Cell Biology

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molecular biology, biochemistry, mass spectrometry, structural biology, high throughput sequencing and bioinformatics. In any single project, many or all of these different approaches may be applied as appropriate and a great strength of the Centre lies in facilitating this process by making advanced technology and expertise available to all groups.

Although our work is investigator led – with individual group leaders free to follow their scientific intuition and curiosity – there are focal themes. One underlying theme in the Centre is the study of how cells establish their polarity, grow, and accurately segregate their chromosomes during division. Studies in this area focus on how cells regulate their growth and division cycles, on the chromosomes themselves, and on the dynamic machinery that accomplishes accurate chromosome segregation during cell division. Closing the circle, this machinery is also critical in determining cell polarity, which can influence the cell cycle and direct the form and fate of daughter cells following division.

Another major group of research projects deals with the regulation of the flow of genetic information from the DNA in the genome into RNA and hence into cellular systems. The synthesis, processing and degradation of RNA lies at the heart of the information processing system of all organisms. We are attempting to understand these important, but highly complex, pathways. Recent analyses in yeast and human cells have cast light on the intimate interconnections between all stages of the gene-expression pathway.

This is an exciting time to be engaged in biological research with dramatic and exciting advances in understanding being made constantly. Much of this

excitement stems from new technology and constant retooling is necessary – both in physical kit and intellectual approaches. However, for researchers like those in the WTCCB who are able to ride the wave, the reward is the huge intellectual satisfaction of knowing that we are playing an important part in the global effort to understand key problems in cell biology, and that this will ultimately underpin revolutions in the understanding and treatment of disease.

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Robin Allshire

Co-workers: Tatsiana Auchynnikava, Pauline Audergon, Harald Berger, Alessia Buscaino, Emilie Castonguy, Sandra Catania, Georgina Hamilton, Erwan Lejeune, Matthew Miell, Alison Pidoux, Manu Shukla, Lakxmi Subramanian, Jitendra Thakur, Nick Toda, Sharon White.

How centromeres are specified: the interplay between heterochromatin, CENP-A chromatin, and kinetochore assembly

Centromeres are the regions on chromosomes where kinetochores assemble; they enable chromosomes to bind spindle microtubules so that during cell division cells receive a complete set of chromosomes.

Most chromosomal DNA is wrapped around nucleosomes, containing core histones H3, H4, H2A and H2B. At the foundation of all kinetochores a centromere specific histone, CENP-A, replaces histone H3, forming CENP-A nucleosomes. This CENP-A chromatin is critical for kinetochore assembly; chromosome segregation is aberrant in cells with defective CENP-A. What determines where on a chromosome CENP-A is assembled instead of histone H3? Primary DNA sequence is not absolute in dictating where active regional centromeres are formed. Instead, ‘epigenetic’ features provide cues that promote the assembly of CENP-A chromatin and the foundations to build kinetochores. Our objective is to understand the nature of these features and how they mediate the replacement of histone H3 with CENP-A to form active centromeres.

We utilize the fission yeast, Schizosaccharomyces pombe, as a model organism. As in human cells, S. pombe centromeres are regional, with repeats surrounding a central domain. ChIP-seq analyses shows that there are on average ~20 CENP-A nucleosomes within the central domain per fission yeast centromere and super resolution PALM imaging indicates the presence of ~74 mEOS2-

CENP-A molecules in each anaphase cluster containing all three centromeres (Figure 1).

We discovered that heterochromatin is required to allow the establishment of CENP-A chromatin and kinetochore proteins. Synthetic heterochromatin made by tethering Clr4 methyltransferase to DNA binding sites bypasses the need for repetitive DNA to form functional centromeres. Currently we are actively pursuing those features of heterochromatin that direct CENP-A chromatin assembly. Our analyses have shown that central CENP-A chromatin is transcribed and defective transcription-coupled chromatin assembly allows promiscuous CENP-A deposition on transcription units. The N-terminal tail of CENP-A is distinct from that of H3 and lacks key post-translational modifications required for retention of H3 nucleosomes during transcription. We propose that heterochromatin constrains RNAPII transcription-coupled events on adjacent centromeric DNA to promote the replacement of H3 with CENP-A.

In the related species, S. octosporus, gene order adjacent to centromeres is preserved but the intervening centromeric DNA retains no detectable homology. ChIP-seq analyses show that CENP-A chromatin associates with these distinct sequences in the same relative location (Figure 2). A conserved transcriptional program from centromeric DNA elements may allow non-coding RNAs to direct centromere formation in a similar manner to that observed in S. pombe.

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Figure 1. ChIP-seq of MNase released nucleosomes showing that a maximum of 20 CENP-A nucleosomes occupy the unique central domain of centromere 2 (left). Super -resolution PALM imaging allows counting of mEOS2-tagged CENP-A molecules in vivo at S. pombe centromere clusters (yellow - right).

Figure 2. Flanking gene order is identical in Schizosaccharomyces pombe and octosporus. Centromeric DNAs share no similarity but ChIP-seq indicates that CENP-ACnp1 is enriched across the intervening region.

Selected Publications: Choi, E.S., Stralfors, A., Castillo, A.G., Durand-Dubief, M., Ekwall, K., and Allshire, R.C. (2011). Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres. J Biol Chem 286, 23600-23607.

Buscaino, A., White, S.A., Houston, D.R., Lejeune, E., Simmer, F., de Lima Alves, F., Diyora, P.T., Urano, T., Bayne, E.H., Rappsilber, J., Allshire R.C. (2012). Raf1 Is a DCAF for the Rik1 DDB1-

like protein and has separable roles in siRNA generation and chromatin modification. PLoS Genet 8, e1002499.

Lando, D., Endesfelder, U., Berger, H., Subramanian, L., Dunne, P.D., McColl, J., Klenerman, D., Carr, A.M., Sauer, M., Allshire, R.C., Heilemann, M., Laue, E.D. (2012). Quantitative single molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast. Open Biology 2,120078.

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A. Jeyaprakash Arulanandam

Co-workers: Bethan Medina, Maria Alba Abad Fernandaz.

Cell division is a fundamental molecular process of life that ensures accurate transfer of genetic information through generations. Errors in cell division often result in daughter cells with inappropriate numbers of chromosomes, a condition associated with cancers and Down’s syndrome. Hence, understanding the molecular mechanisms of cell division is crucial in order to make an impact on human health.

To achieve high fidelity genetic transfer, cell division involves highly orchestrated events such as chromosome condensation, attachment of microtubules (MTs) to kinetochores (KTs), formation of a stable spindle assembly, movement of sister chromatids to opposite poles and the successful completion of cytokinesis. Such a complicated network of events that takes place between the onset and exit of mitosis is regulated by a number of mitotic molecular machines (including condensin, cohesin, Chromosomal Passenger Complex (CPC), KMN (Knl1-Mis12-Ndc80) network, Ska complex, spindle checkpoint and the anaphase promoting complex) involving an extensive network of protein-protein interactions.

Among the key molecular interactions, those involving the Chromosomal Passenger Complex (CPC) and the Ska complex are currently being investigated in our lab, mainly due to their essential role in KT-MT attachments and timely mitotic progression. While the CPC, with its enzymatic core Aurora B, orchestrates various mitotic events from chromosome condensation to cytokinesis by

phosphorylating the right substrate at the right place and time, the Ska complex physically couples chromosomes to the spindle MTs by interacting with KT associated proteins and is implicated in the timely onset of anaphase.

Understanding the protein interactions involving essential regulators such as the CPC and the Ska complex is crucial in understanding how networks of protein-protein interactions are translated into mitotic regulation and thus in unraveling the molecular mechanisms of accurate cell division. Towards this goal, we take interdisciplinary approach combining structural, biochemical and cell biological methods. We use molecular biology and biochemical methods to characterize protein interactions in vitro, X-ray crystallography, SAXS and negative staining EM for structural analysis and in vitro or/and in vivo functional assays using structure based mutants for functional characterization. The structural and functional insights that we would obtain from these studies will also allow us to explore possibilities of targeting specific mitotic regulators in fighting cancer.

Structural Biology of Cell Division

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Selected Publications: Jeyaprakash, A. A., Santamaria, A., Jayachandran, U., Chan, Y, W., Benda, C., Nigg, E. A., and Conti, E. (2012). Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface. Mol Cell 46, 274-286.

Chan, Y. W., Jeyaprakash, A. A., Nigg, E. A., and Santamaria, A. (2012). Aurora B controls kinetochore-microtubule attachments by

Figure 2. Structural assembly of the Ska complex. Top panel: Crystal structure of the Ska core complex as seen in the crystal structure. Bottom panel: Over all model for the full length Ska complex.

inhibiting Ska complex-KMN network interaction. J Cell Biol 196, 563-571.

Jeyaprakash, A.A., Basquin, C., Jayachandran, U., and Conti, E. (2011). Structural basis for the recognition of phosphorylated histone H3 by the surviving subunit of the chromosomal passenger complex. Structure 19, 1625-1634.

Figure 1. Crystal structure of the CPC core complex.

Figure 3. Structural insight into the role of the Ska complex in stabilising KT-MT interaction.

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The mechanism and machinery of RNA splicing are highly conserved throughout eukaryotes, and the budding yeast Saccharomyces cerevisiae makes an excellent model system, permitting the application of genetic approaches in parallel with molecular studies. In addition to investigating the functions and molecular interactions of yeast splicing factors, we are interested in links between RNA splicing and other metabolic processes, especially transcription. Our approaches include: biochemical analyses of splicing in vitro, quantitative RT-PCR, chromatin immunoprecipitation, in vivo RNA-protein cross-linking and molecular genetics.

Eight RNA helicases regulate conformational changes during the cycle of spliceosome assembly, catalysis and disassembly. Despite extensive analysis, little is known about their substrates and mechanism of action or how the helicases themselves are regulated. Amongst these, Brr2p has an unusual architecture, with two helicase modules. Only the amino-terminal helicase module is catalytically active, and the role of the carboxy-terminal module is unclear. Combining genetic and biochemical approaches, we showed that the C-terminus of Brr2p interacts physically and genetically with other spliceosomal RNA helicases, including Prp2p and Prp16p in vivo. In vitro, the C-terminal domain alters the ATPase activities of both Prp2p and Prp16p. These interactions depend on the ATP/ADP state of Brr2p and are also affected by the presence of RNA. We propose that the second helicase module of Brr2p evolved

Jean Beggs

Co-workers: Ross Alexander, David Barrass, Keerthi Chathoth, Olivier Cordin, Vanessa Cristão, Amit Gautam, Eve Hartswood, Daniela Hahn, Steve Innocente, Jane Reid.

as a protein-interaction domain that controls the activities of multiple splicing helicases at the catalytic centre of the spliceosome (Figure 1; Olivier Cordon and Daniela Hahn). In addition, UV cross-linking combined with high throughput sequencing (CRAC) analyses identified the RNA interaction partners of Brr2 and the sites of interaction within these RNAs. This has provided insight into the mechanism of U4/U6 unwinding by Brr2. Additionally, CRAC analysis with a mutant Brr2 protein revealed a novel role for Brr2, driving conformational rearrangements within the spliceosome between the two steps of splicing, helping to reposition the substrate RNA in the catalytic centre for the second splicing reaction (Daniela Hahn; collaboration with the Tollervey group).

A major interest is analysis of the functional coupling of transcription and splicing, following on our discovery that RNA polymerase II pauses transiently on intron-containing genes in a splicing-dependent manner (Alexander et al., 2010). We have identified two candidate coupling factors. These are splicing factors that, when mutated, affect transcription elongation across introns. We speculate that there may be multiple transcriptional checkpoints that may be linked to checking the fidelity of the splicing process when it occurs co-transcriptionally (Ross Alexander, David Barrass, Keerthi Chathoth, Steve Innocente, Jane Reid).

The mechanism and regulation of pre-mRNA splicing and links to transcription

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Figure 1. A model for the function of Brr2p driving conformational rearrangements in the spliceosome, and as a regulator of other splicing helicases. Numbers indicate different stages of the splicing cycle. The positions of the various helicases indicate the stage of spliceosome progression for which each is required. Solid lines radiating from Brr2p indicate a stimulatory or inhibitory effect on the ATPase activities of the connected helicases. An asterisk beside a helicases name indicates the pre-ATP hydrolysis state of the protein.

Selected Publications: Alexander, R., Innocente, S., Barrass, J.D. and Beggs, J.D. (2010) Splicing causes RNA polymerase to pause in yeast. Mol Cell 40,582-593.

Kudla, G., Granneman, S., Hahn, D., Beggs, J.D. and Tollervey, D. (2011) Mapping in vivo RNA-RNA interactions by crosslinking, ligation and sequencing of hybrids. PNAS 108, 10010-10015. doi 10.1073/PNAS.1017386108.

Weber, G., Cristão, V.F., Alves, F.deL., Santos, K.F., Holton, N., Rappsilber, J., Beggs, J.D. and Wahl, M.C. (2011) Molecular Mechanism for the Function of Aar2p as an Assembly Chaperone for U5 snRNP. Genes Dev. 25, 1601-1612. PMID: 21764848.

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DNA methylation and CpG islands

We study the biological significance of genomic DNA modification, which occurs at the short dinucleotide 5’CpG. In most of the genome the CpGs are under-represented and predominantly methylated. Discrete genomic regions near gene promoters, however, display a CpG frequency that is ~10-fold higher than elsewhere. These so-called “CpG islands” (CGIs) are associated with about 60% of protein-coding genes, but we recently showed that many mark novel promoters of unknown function. Unlike the bulk genome, CGIs are usually methylation-free. In fact transcription of the associated gene is invariably silenced if they become methylated either naturally or as a result of disease (e.g. cancer).

The functional significance of CGIs was until recently obscure. In 2010, however, we showed that a protein with a CXXC domain, Cfp1, binds to clustered non-methylated CpGs and facilitates recruitment of trimethylation at lysine 4 of histone H3 (H3K4me3) - a signature chromatin mark at CGIs. Therefore shared features of CGI DNA sequences attract enzymes that create a chromatin structure that is typical of active promoters (Figure 1). We recently extended this work through study of Cfp1-deficient embryonic stem cells. By expressing a form of Cfp1 that cannot bind to CpG, we were able to show that DNA binding restricts H3K4me3 to CGI promoters. In its absence, this mark accumulates at other gene regulatory regions, affecting the expression of local genes.

Our group also studies a human autism spectrum disorder called Rett syndrome, which is caused by mutations in the X-linked MECP2 gene. Rett syndrome has been thought of as a “neurodevelopmental disorder”, implying that defects arise during early development. We previously showed in an animal model of the condition that severe symptoms could be reversed in adults, casting doubt on their developmental origin. Extending this finding, we showed in collaboration with Drs Stuart Cobb (Glasgow University) and Gernot Riedel (Aberdeen University) that reversal is accompanied by restoration of normal morphology to neurons and by improvements in both breathing and behaviour. In addition, we recently discovered that inactivation of the Mecp2 gene in adult animals of various ages caused onset of severe symptoms, thus proving that there is a lifelong need for the MeCP2 protein. Based on our evidence for reversibility, we are currently exploring potential therapeutic approaches to this profound condition.

Selected Publications: Robinson, L., Guy, J., McKay, L., Brockett, E., Spike, R.C., Selfridge, J., De Sousa, D., Merusi, C., Riedel, G., Bird, A., Cobb, S.R. (2012). Morphological and functional reversal of phenotypes in a mouse model of Rett syndrome. Brain. 2012 Apr 23. [Epub ahead of print].

Cheval, H., Guy, J., Merusi, C., De Sousa, D., Selfridge, J., and Bird, A. (2012). Postnatal inactivation reveals enhanced requirement for MeCP2 at distinct age windows. Hum Mol Genet. 2012 Jun 13. [Epub ahead of print].

Thomson, J.P., Skene, P.J., Selfridge, J., Clouaire, T., Guy, J., Webb, S., Kerr, A.R., Deaton, A., Andrews, R., James, K.D., Turner, D. J., Illingworth, R., Bird, A. (2010). CpG islands influence chromatin structure via the CpG-binding protein Cfp1. Nature 464, 1082-1086.

Adrian Bird

Co-workers: Kyla Brown, Helene Cheval, Justyna Cholewa-Waclaw, Thomas Clouaire, Dina De Sousa, Robert Ekiert, Jacky Guy, Martha Koerner, Sabine Lagger, Matthew Lyst, Cara Merusi, Gabriele Schweikert, Jim Selfridge, Christine Struthers, Elisabeth Wachter.

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Figure 1. The chromatin state at CpG islands. A. CGIs usually exist in an unmethylated transcriptionally permissive state. They are marked by histone acetylation (H3/H4Ac), H3K4 trimethylation (recruited by the CG binding protein Cfp1) and depletion of H3K36 trimethylation (directed by the CG binding protein Kdm2a). Nucleosome deficiency and constitutive binding of RNAPII may also contribute to this transcriptionally permissive state. B. DNA methylation is associated with stable long-term silencing of CGI promoters. This can be mediated by methyl-binding domain (MBD) proteins, which recruit co-repressor complexes associated with histone deacetylase (HDAC) activity, or may be due to direct inhibition of transcription factor binding by DNA methylation. C. CGIs are often silenced by polycomb group proteins and may be involved in their recruitment. An unknown CGI-binding factor could be responsible for recruiting PRC2 to CGIs leading to trimethylation of H3K27 and consequent recruitment of PRC1 complexes. Note that the transcriptionally permissive and polycomb repressed states can co-exist at bivalent CGIs, predominantly in totipotent embryonic cells.

Transcriptionally permissive

Repressed by DNA methylation

RNAPII

TFsKdm2a Cfp1

Setd1

CpG island

Repressed by Polycomb

PRC2

? PRC1

Unmethylated CpG

Methylated CpG

H3K36me2

H3K4me3

H3/H4Ac

H3K27me3

Transcription factorMethyl-binding domain protein

Histone deacetylase

Unknown Polycomb recruiter

HDAC

MBD XTFs

Deaton_Figure 4

Transcriptionally permissive

Repressed by DNA methylation

RNAPII

TFsKdm2a Cfp1

Setd1

CpG island

Repressed by Polycomb

PRC2

? PRC1

Unmethylated CpG

Methylated CpG

H3K36me2

H3K4me3

H3/H4Ac

H3K27me3

Transcription factorMethyl-binding domain protein

Histone deacetylase

Unknown Polycomb recruiter

HDAC

MBD XTFs

Deaton_Figure 4

A

B

C

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Atlanta Cook

Co-workers: Uma Jayachandran, Urszula Wolkowicz.

Translation is the central process in biology during which genetic information encoded on mRNAs is read by the ribosome and polypeptides are synthesized. Extensive biochemical studies on prokaryotic ribosomes have given insights into the assembly of this machine, while structural studies have illuminated its workings during translation. In eukaryotic ribosomes, two areas where we lack a good mechanistic understanding are in ribosome biogenesis and translational control.

Eukaryotic ribosome biogenesis is vastly more complex than in prokaryotes, requiring more than 200 additional factors and proceeding through multiple cellular compartments. The process centres around the transcription of a long ribosomal RNA transcript in the nucleolus. Chemical modification of bases and the association of small subunit ribosomal proteins occur at this early stage. A series of RNA cleavage events then separates the pre-40S and pre-60S particles, which exit the nucleus as almost fully assembled pre-ribosomal particles. These particles associate with late-acting biogenesis factors that also act as transport adaptors, mediating interactions with the nucleocytoplasmic transport machinery. Final maturation is completed in the cytoplasm where late-acting biogenesis factors are removed and the mature ribosomal particles can associate on mRNAs. Nucleo-cytoplasmic transport is a critical but poorly understood step in maturation. I am interested in understanding the mechanistic roles that late-acting

ribosomal biogenesis factors play in transport and maturation.

The rate of translation of a given mRNA depends on both its stability and its availability for translation. The association of regulatory protein complexes with distinct elements in the 3’UTRs of cognate mRNAs can have a major impact on the fate of these mRNPs. Some regulatory proteins can stabilize or destabilize mRNPs by protecting them from or recruiting them to the RNA degradation machinery. The association of regulatory proteins with translating mRNAs can also affect how successfully these mRNAs recruit ribosomes (translational control). In addition to their roles in regulating cellular mRNAs, some protein complexes involved in translational control have been implicated as host factors during the life cycle of several plus-stranded RNA viruses. These viruses have a single-stranded RNA genomes that resemble native mRNAs. How these viruses use 3’-UTR binding proteins from the host to promote their replication is not well understood.

We use structural biology (primarily using X-ray crystallography) to gain functional insights into proteins and macromolecular complexes involved in both eukaryotic ribosome biogenesis and translational control.

Structural biology of macromolecular complexes involved in RNA metabolism

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Selected Publications: Bono F, Cook AG, Grünwald M, Ebert J, Conti E. (2010) Nuclear import mechanism of the EJC component Mago-Y14 revealed by structural studies of importin 13. Mol Cell. 37:211-22.

Cook AG, Fukuhara N, Jinek M, Conti E. (2009) Structures of the tRNA export factor in the nuclear and cytosolic states. Nature. 461:60-5.

Wolkowicz, U. M., Cook, AG. NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold. Nucleic Acids Research 2012; doi: 10.1093/nar/gks696.

Figure 1A. Association of regulatory proteins to the 3’UTR of regulated mRNAs can affect translation in different ways, either through degradation or promoting/interfering with translation initiation. B. Structure of the dimerization domain of NF90/NF45, a protein complex thought to be involved in translational control. C. NF90 has two dsRNA binding domains (dsRBDs) that were not present in the structural analysis. Based on the structure and using coimmunoprecipitation studies, we have demonstrated that NF45 dimerizes with two other proteins, SPNR and Zfr, forming a variety of RNA recognition platforms.

A B

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William C. Earnshaw

Co-workers: Helena Barysz, Dan Booth, Mar Carmena, Florence Gohard, Alexander Kagansky, Nuno Martins, Oscar Molina, Hiromi Ogawa, Shinya Ohta, Diana Papini, Melpi Platan, Itaru Samejima, Kumiko Samejima, Paola Vagnarelli, Giulia Vargiu, Laura Wood.

Our studies aim to answer the following three questions: How do mitotic chromosomes form and segregate in mitosis? What is the chromatin environment of the centromere that provides an epigenetic landscape permissive for kinetochore assembly? How does the chromosomal passenger complex (CPC) regulate mitotic events?

At the end of G2 phase, interphase chromatin undergoes a dramatic change as individual chromatids form from the amorphous chromosome territories of interphase nuclei. This process involves only a 2-3-fold compaction of the chromatin, but requires a profound reorganization of the chromatin fibre. The basis of this reorganisation is still mysterious despite many decades of intense study. Non-histone proteins are now widely believed to have essential roles in mitotic chromosome formation and architecture. In an ongoing collaboration with Juri Rappsilber, we use Multi-Classifier Combinatorial Proteomics (MCCP) to determine how depleting key members of important protein complexes affects the global chromosome proteome as well as that at the kinetochore. To date most is known about the condensin I and II complexes, which are proposed to be involved in shaping chromosomes. However vertebrate mitotic chromosomes can form and segregate in the absence of condensins. We have found that the abundant chromosome-associated chromokinesin, KIF4, is also required for chromosomes to form a robust architecture. KIF4 cooperates with condensin, and the

two are interdependent for their efficient association with mitotic chromosomes. Surprisingly, DNA topoisomerase IIα appears to function in opposition to the condensin/KIF4 pathways and knockdown of topo II can rescue several of the chromosomal phenotypes associated with the loss of condensin or KIF4.

We are using both a human synthetic artificial chromosome and the natural unique centromeres of several chicken chromosomes to map the histone modifications that render chromatin permissive for kinetochore assembly. These studies reveal that centromere chromatin is surprisingly plastic, and that keeping a critical balance of transcription (not too much and not too little) is essential for kinetochore maintenance.

Other studies have analysed functional interactions between the CPC and the mitotic kinase Polo. By combining genetic, biochemical and advanced microscopy studies, we found that Aurora B activates Polo kinase at kinetochores during prophase in Drosophila and human cells. This is particularly interesting because the two kinases have opposite effects. Polo promotes microtubule binding at kinetochores whilst Aurora B promotes microtubule release. We hypothesize that INCENP integrates and coordinates the activity of these two kinases, thereby enabling accurate chromosome segregation.

The role of non-histone proteins in chromosome structure and function during mitosis

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Selected Publications: Ohta S., J.-C. Bukowski-Wills., L. Sanchez-Pulido, F. de L. Alves, L. Wood, Z.A. Chen, M. Platani, L. Fischer, D.F. Hudson, C.P. Ponting, T. Fukagawa, W.C. Earnshaw* and J. Rappsilber*. (2010). The protein composition of mitotic chromosomes determined using multi-classifier combinatorial proteomics. CELL 142: 810-821.

Vagnarelli P., S. Ribeiro, L. Sennels, L. Sanchez-Pulido, F. de L. Alves, T. Verheyen, D.A. Kelly, C.P. Ponting, J. Rappsilber and

W.C. Earnshaw. (2011). Repo-Man coordinates chromosomal reorganisation with nuclear envelope reassembly during mitotic exit. DEV. CELL 21: 328-342.

Carmena M., X.Pinson, M. Platani, Z. Salloum, Z. Xu, A. Clark, F. MacIsaac, H. Ogawa, U. Eggert, D.M. Glover, V. Archambault, W.C. Earnshaw. (2012). The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres. PLoS Biol. 10, e1001250.

Figure 1A. KIF4, Condensin and Topoisomerase IIα all colocalise on mitotic chromosomes. B. Mitotic chromosomes lacking KIF4 plus condensin look much worse than chromosomes lacking either one alone. C. Model for the action of KIF4, condensin and topoisomerase IIα in shaping mitotic chromosomes.

B C

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We study how the cell biology of mitosis is co-ordinated with cell cycle progression. The spindle checkpoint normally prevents cells with spindle or kinetochore defects from initiating chromosome segregation. Mutations in the mad (mitotic arrest defective) or bub (budding uninhibited by benomyl) genes inactivate the checkpoint and allow cells with defective spindles to proceed through mitosis. Such cell divisions lead to inaccurate chromosome segregation, aneuploidy and death.

A single unattached kinetochore is sufficient to activate the spindle checkpoint, and we are particularly interested in how such kinetochores generate signals that delay anaphase onset throughout the mitotic apparatus. Mad1 recruits and activates Mad2 at kinetochores, and Bub1 recruits Mad3 (Fig.1A). Once activated, Mad3 and Mad2 interact with Cdc20 to form the mitotic checkpoint complex (MCC) which is crucial for inhibition of the anaphase-promoting complex (APC/C), and thereby delays anaphase onset.

A major focus of our current research is to identify relevant substrates of the Mph1, Bub1 and Aurora (Ark1) kinases. Mph1 and Ark1 kinase activities are critical for spindle checkpoint arrest and we have identified several important substrates. The kinetochore protein Spc7/KNL1 is phosphorylated by both kinases, and when conserved MELT motifs are phosphorylated by Mph1 they become a binding site for the Bub3-Bub1 checkpoint complex (Fig.1A-C). In addition, Mph1 kinase phosphorylates

Mad2 and Mad3, and their modification is necessary to form stable MCC-APC/C complexes and thereby maintain spindle checkpoint arrests (Fig.1D).

One all chromosomes are bioriented on the spindle, the checkpoint is satisfied. We have identified Spc7 as a key recruitment factor for protein phosphatase 1 (PP1)-mediated checkpoint silencing. Mutation of highly conserved PP1 binding motifs leads to very inefficient checkpoint silencing and a lethal metaphase arrest.

Selected Publications: Shepperd, L.A., Meadows, J.C., Sochaj, A.M., Lancaster, T.C., Zou, J., Buttrick, G.J., Rappsilber, J., Hardwick, K.G. and Millar, J.B.A. (2012). Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle checkpoint. Current Biology, 22, 891-199.

Zich, J., Sochaj, A.M., Syred, H.M., Milne, L., Cook, A.G., Ohkura, H., Rappsilber, J., and Hardwick, K.G. (2012). Kinase activity of fission yeast Mps1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex. Current Biology, 22, 296-301.

Meadows, J.C., Shepperd, L.A., Vanoosthuyse, V., Lancaster, T.C., Sochaj, A.M., Buttrick, G.J., Hardwick, K.G., and Millar, J.B.A. (2011) Spindle checkpoint silencing requires association of PP1 to both Spc7 and Kinesin-8 motors. Developmental Cell, 20, 739-750.

Kevin G. Hardwick

Co-workers: Karen May, Kostas Paraskevopoulos, Sjaak van der Sar, Onur Sen, Alicja Sochaj, Ivan Yuan, Judith Zich.

Spindle checkpoint activation and silencing mechanisms

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Figure 1A. Model for spindle checkpoint protein recruitment to kinetochores. B. Mph1 kinase phosphorylates Spc7 in vitro on conserved MELT motifs. C. Spc7 phosphomimic mutants (T9E) efficiently bind Bub3-Bub1 complexes in vitro and are sufficient to recruit them to kinetochores. Recombinant MBP fusion proteins were coupled to beads and then used to pull Bub complexes out of yeast extracts. D. Mph1 kinase activity is also required for stable binding of Mad2 and Mad3 to the APC/C. Cells were released into mitosis (from a G2 block) and then challenged with an anti-microtubule drug (carbendazim). The APC/C was purified from extracts (using Apc4-TAP) and analysed for associated checkpoint proteins by immunoblotting.

A

D

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20

Lea A. Harrington

Co-workers: Larissa Christian, Jennifer Dorrens, Laura Gardano, Fiona Pryde, Fabio Pucci, Elisa LH Wong.

Selected Publications: Taboski, M.A.S., Sealey, D.C.F., Dorrens, J., Tayade, C., Betts, D.H., and Harrington, L. (2012). Long telomeres bypass the requirement for long telomeres in human tumorigenesis. Cell Reports 1, 91-98.

Gardano, L., Holland, L., Oulton, R., Le Bihan, T., and Harrington, L. (2012). Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic Acids Res Mar;40(5):e36.

Sealey, D.C., Kostic, A.D., LeBel, C., Pryde, F., and Harrington, L. (2011). The TPR-containing domain within Est1 homologs exhibits species-specific roles in telomerase interaction and telomere length homeostasis. BMC Mol Biol 12, 45.

All eukaryotes that possess linear chromosomes require a mechanism to maintain a reserve of non-coding DNA at the extreme chromosomal terminus, called the telomere. Depletion of telomere DNA during cell division, a natural consequence of genome replication, eventually curtails the viability and proliferation of normal and cancerous cells. Telomere depletion is mitigated by telomerase, an enzyme that is comprised of two essential subunits: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TERC) that provides the template for new telomere DNA synthesis. In recent years, our research has sought to address the impact of partial or complete loss of telomerase (TERT) function in a physiological context that more closely resembles that of human stem cells; that is, when telomeres are initially longer. In genetic model systems, we have uncovered distinct cellular responses to partial or complete loss of telomerase function that vary from induction of alternate modes of telomere maintenance (such as recombination), to a compensatory increase in telomere length in cells that retain low levels of telomerase activity. We are also interested in the cell signaling mechanisms that are induced by critically short telomeres, alone or when combined with other genotoxic insults such as irradiation and DNA damage. Taken together, we aim to uncover mechanisms by which modulation of telomerase function can be used to alleviate age-associated diseases that may be exacerbated by loss of telomere function.

Molecular mechanisms of telomerase function and telomereintegrity in vivo

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Figure 1. Human telomerase expression at yeast telomeres can induce senescence in S.cerevisiae cells. TOP: Active human telomerase causes growth arrest with a 2-fold increase in yeast cell doubling time. BOTTOM: Expression of telomeric DNA binding-deficient human telomerase and inactive human telomerase abolished the growth inhibition, confirming that this senescence is a potential readout for human telomerase function. E. L. H. Wong, L. Harrington, unpublished.

Figure 2. LEFT: Lengthening of telomeres occurs in telomerase negative mouse embryonic fibroblasts (MEF) when expressing the Retinoblastoma-like 2 (Rbl2) protein. Black arrow indicates average telomere length. RIGHT: Cells expressing Rbl2 exhibit a change (Note: shift to left) in the telomere signal ratio between short (P) and long (Q) arms of chromosome. F. Pucci, L. Harrington, unpublished.

22

Adele Marston

Co-workers: Julie Blyth, Colette Connor, Eris Duro, Josefin Fernius, Stefan Galander, Olga Nerusheva, Claudia Schaffner, Kitty Verzjilbergen, Nadine Vincenten.

We are studying the molecular mechanisms that ensure the accurate segregation of chromosomes during cell division. Errors in chromosome segregation generate daughter cells with the wrong number of chromosomes, known as aneuploidy, and this is associated with cancer, birth defects and infertility. To uncover conserved and fundamental mechanisms of chromosome segregation we employ the distantly related budding and fission yeasts as model systems.

The partitioning of the genetic material into daughter cells begins with the duplication of the chromosomes. Coupled to the synthesis of an exact copy of the DNA is the assembly of protein complexes that are important for chromosome structure and organization. One such protein complex is the cohesin complex that holds the two newly duplicated chromosomes together. For accurate chromosome segregation during mitosis, the sister chromatids must capture microtubules from opposite poles. The kinetochore assembles on the centromere of each chromosome to connect them to microtubules. Cohesin resists the pulling forces of microtubules to allow all the chromosomes to make proper bipolar attachments. Once this has occurred, cohesin is abruptly lost, triggering the movement of identical sister chromatids to opposite poles. Cohesin is present along the chromosomes but is most highly enriched in a large domain surrounding the centromere, known as the pericentromere.

Our overall goal is to understand the composition and function of the pericentromere. Work currently underway in the lab focuses on three overlapping questions.

1. How is cohesin established in the pericentromere?We identified a subcomplex in the budding yeast kinetochore that is important for cohesin enrichment in the pericentromere. We are currently investigating the mechanism of cohesin loading at the centromere in budding yeast.

2. How does the pericentromere regulate the onset of anaphase? A pericentromeric protein, Shugoshin, functions in a surveillance mechanism that prevents anaphase onset until all sister chromatids have made proper bipolar attachments. We aim to understand the role of Shugoshin in sensing the lack of tension associated with incorrect attachments and in signalling these errors to the cell cycle machinery.

3. How is the pericentromere modified for meiosis?Meiosis is a modified cell division that produces gametes through two consecutive rounds of chromosome segregation. During the first meiotic division, the configuration of the chromosomes must change compared to mitosis because it is the maternal and paternal chromosomes that are segregated, rather than the sister chromatids. Our goal is to identify these changes through proteomic and genomic screens

The Role of the Pericentromere during Mitosis and Meiosis

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Figure 1. Time lapse imaging of Sgo1 localization as cells progress from G1 into a metaphase arrest. Sgo1-GFP and Mtw1-tdTomato (kinetochores) are shown in green and red, respectively, in the merged image.

Selected Publications: Bizzari, F. and Marston, A.L. (2011) Cdc55 coordinates spindle assembly and chromosome disjunction during meiosis. J Cell Biol. 193, (7)

Clift, D. and Marston, A.L. (2011) The role of Shugoshin in meiotic chromosome segregation. Cytogenet Genome Res. 133, 234-42.

Fernius, J. and Marston, A.L. (2009) Establishment of cohesion at the pericentromere by the Ctf19 kinetochore subcomplex and the replication fork-associated factor, Csm3. PLoS Genet. e1000629.

Figure 2. Schematic diagram showing the relative localization of Sgo1 and the Bub1 kinase that is required for its localization.

Figure 3. Schematic diagram illustrating the effect of spindle tension on Sgo1 localization.

24

Gracjan Michlewski

Co-workers: Nila Roy Choudhury, Jakub Nowak.

Regulation of microRNA biogenesis and function

MicroRNAs (miRNAs) constitute a large family of short (21-23nt) non-coding RNAs that regulate protein production and control a variety of biological processes. Given their diverse functions it is not surprising that their spatial and temporal expression is tightly regulated. MiRNAs are derived from primary transcripts (pri-miRNA) by sequential nuclear and cytoplasmic processing events. In the nucleus, RNase Drosha and DGCR8 generate stem-loop precursors (pre-miRNA), which are exported to the cytoplasm. In the cytoplasm, RNase Dicer cleaves the pre-miRNA leaving miRNA duplex. Subsequently, one strand of this duplex is incorporated into RNA-Silencing Complex (RISC), which targets partially complementary mRNAs and controls their expression. It has been established that the multifunctional RNA binding protein hnRNP A1 is required for the processing of miR-18a at the nuclear step of Drosha cleavage. This suggested a previously uncharacterized role for general RNA binding proteins as auxiliary factors that alter the biogenesis of specific miRNAs.

At present, around 1500 miRNAs are annotated in the human genome. Each of them has the capacity to regulate several, possibly hundreds, of mRNA targets. In spite of the great effort to understand various biological roles of individual miRNAs, very little has been done to unravel the regulation of their own biogenesis. Until now only a handful of miRNAs have been investigated individually for the intrinsic features and trans-acting factors that play significant roles in their production. Our hypothesis is that

regulation of miRNA biogenesis pathways in mammalian cells is widespread, allowing for rapid and global adaptation of the gene expression machinery to internal and external stimuli.

By combining a wide spectrum of molecular biology and biochemistry techniques, including functional assays in human and mouse cultured cells, cellular models of neuronal differentiation, RNA deep sequencing, RNA chromatography combined with SILAC Mass Spectrometry, RNA structure probing and in vitro processing assays, we are dissecting fine details of miRNA biogenesis.

Given diverse functions of miRNAs, it is not surprising that aberrant miRNA expression is linked to a variety of human pathological states including initiation, progression and metastasis of human cancers. Importantly, miRNAs are seen as promising tools for rational therapeutic approaches, thus thorough knowledge about their biogenesis pathways will be instrumental in understanding miRNA cellular functions and in designing effective miRNA-based therapies.

Selected publications: Michlewski, G., Guil, S., Semple, C.A., and Caceres, J.F. (2008). Posttranscriptional regulation of miRNAs harboring conserved terminal loops. Mol Cell 32, 383-393.

Michlewski, G., and Caceres, J.F. (2010). Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis. Nat Struct Mol Biol 17, 1011-1018.

Michlewski, G., and Caceres, J.F. (2010). RNase-assisted RNA chromatography. RNA 16, 1673-1678.

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Figure 1. Multiple sequence alignments for the human genome with 16 other vertebrate genomes show very high level of conservation in the terminal loop regions of some human pri-miRNAs. (Upper panel) Conservation across pri-miR-18a corresponds to a persistently high phastCons conservation profile across stem and terminal loop region. (Lower Panel) Conservation across pri-miR-27a includes a clear dip in phastCons conservation profile corresponding to the terminal loop region.

Figure 2. The conserved terminal loop of pri-miR-18a is required for its efficient cleavage by Drosha. (Left panel) Validated secondary structures of wild-type and terminal loop mutants of pri-miR-18a. (Right panel) In vitro processing of wild-type pri-miR-18a and loop mutants is shown.

26

Hiro Ohkura

Co-workers: Sally Beard, Manuel Breuer, Sara Clohisey, Nathalie Colombié, Fiona Cullen, Agata Gɫuszek, Heather Grey, Karolina Mateja, Elvira Nikalayevich, Heather Syred.

Accurate segregation of chromosomal DNA is essential for life. A failure or error in this process during somatic divisions could result in cell death or aneuploidy.

Furthermore, chromosome segregation in oocytes is error prone in humans, and mis-segregation is a major cause of infertility, miscarriages and birth defects. The chromosome segregation machinery in oocytes shares many similarities with these in somatic divisions, but also has notable differences. The differences include clustering of meiotic chromosomes in the nucleus and spindle formation without centrosomes in oocytes. In spite of its importance for human health, little is known about the molecular pathways which set up the chromosome segregation machinery in oocytes. Defining these molecular pathways is crucial to understand error-prone chromosome segregation in human oocytes. Furthermore, evidence indicates that these apparent oocyte-specific pathways also operate in mitosis, although less prominently, to ensure the accuracy of chromosome segregation. Therefore uncovering the molecular basis of these pathways is also important to understand how somatic cells avoid chromosome instability, a contributing factor for cancer development.

To understand the molecular pathways which set up the chromosome segregation machinery in oocytes, we take advantage of Drosophila oocytes as a “discovery platform” because of their similarity to mammalian oocytes and suitability to a genetics-led mechanistic analysis. In Drosophila oocytes, like in human oocytes,

meiotic chromosomes form a compact cluster called the karyosome within the nucleus. Later, meiotic chromosomes assemble the spindle without centrosomes, establish bipolar attachment and congress within the spindle. We have isolated a number of Drosophila mutants which are defective in both chromosome organisation and spindle formation in oocytes. We identified two conserved protein kinases (NHK-1 and SRPK) involved in karyosome formation, and showed phosphorylation of BAF, a linker between nuclear envelope and chromatin is essential to release chromatin from the nuclear envelope. We also found that SRPK is essential for spindle microtubule assembly in oocytes.

In parallele, we apply ‘omics approaches to understand microtubule regulation in oocytes, and in other dividing and non-dividing cells. We found that the gamma-tubulin recruiting complex Augmin is important for chromosome congression, but not bulk microtubule assembly, in oocytes. Further identification of microtubule-associated proteins revealed novel regulations and functional redundancies. In summary, chromosomal proteins, microtubule-associated proteins and protein kinases work together to establish meiotic chromosome organisation and the spindle in oocytes.

Meiotic cell division and microtubule regulation

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Selected Publications: Meireles, A. M., Fisher, K. H., Colombié, N., Wakefield, J. G., and Ohkura, H. (2009). Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis. J. Cell. Biol. 184, 777-784.

Lancaster, O. M., Breuer, M., Cullen, C. F., Ito, T., and Ohkura, H. (2010). The meiotic recombination checkpoint suppresses

NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila. PLoS Genet. 6, e1001179.

Loh, B. J., Cullen, C. F., Vogt, N., and Ohkura, H. (2012). The conserved kinase SRPK regulates karyosome formation and spindle microtubule assembly in Drosophila oocytes. J. Cell Sci. 125.

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Figure 4. SRPK regulates karyosome formation and spindle microtubule assembly in oocytes.

Figure 1. The conserved kinase SRPK is important for karyosome formation.

Figure 2. The conserved kinase SRPK is essential for clustering of heterochromatin (highlighted by HP1) in oocytes.

Figure 3. SRPK is essential for spindle microtubule assembly in oocytes.

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Juri Rappsilber

Co-workers: Adam Belsom, Jimi-Carlo Bukowski-Wills, Zhuo Angel Chen, Colin Combe,Flavia de Lima Alves, Cristina Furlan, Lutz Fischer, Georg Kustatscher, Paul Mason, Lauri Peil, Michaela Spitzer, Salman Tahir, Sakharam Waghmare, Karen Wills, Juan Zou.

We aim to advance our understanding of the structure and function of chromatin by describing in vivo protein-protein interactions in large scale. This year we have conducted a number of cross-linking analyses to define structural details of multi-protein complexes. The speed of such analyses was enhanced vastly by the maturity of our graphical user interface which provides access to our in-house mass spectrometric data analysis pipeline Xi, developed by our lab for identification of cross-link sites. Our analysis of a static, 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF has established cross-linking/mass spectrometry (3D proteomics) as an integrated structure analysis tool for large multi-protein complexes (Chen et al., EMBO J. 2010). We now add to this tool box the ability to visualise data in a highly integrative and interactive web interface. This provides the basis for intuitive data interrogation and rapid hypothesis generation. We developed a spectrum viewer that surpasses any previous attempt to allow scientists to challenge the automated matching of a mass spectrum with a peptide from a protein database. All linkage data are then visualised in a protein viewer that uses newly developed visualisation concepts for network data. Having site resolution we added protein stick representation to the canonical protein network graphs, again in an interactive web tool. The spectrum viewer is already available open source and may change the way biologists at large interact with mass spectrometric data, e.g. by providing on-line access to mass spectrometric evidence for protein modification sites.

Defining the protein composition of mitotic chromosomes in collaboration with the Earnshaw lab (Ohta et al., Cell 2010), we noticed a large number of hitchhiker proteins, highly unexpected proteins found in our study, associated with chromosomes. This threw up a number of questions. Did lysis destroy a finely tuned balance or unknown mechanism that exists within mitotic cells and protects mitotic chromosomes from extensive association of cytoplasmic proteins following the breakdown of the nuclear envelope at the end of G2? Or do, in fact, mitotic chromosomes already sequester soluble proteins in the cell merely because they can (due to the surface area and physico-chemical properties of mitotic chromosomes and the access to soluble proteins in the absence of the nuclear envelope)? In the latter case, what would happen to these proteins at the end of mitosis, when the nuclear envelope reforms? Would they be excluded, or trapped in the nucleus and subsequently removed (exported/degraded), or trapped and remain stuck, possibly invisible to the cell? In the latter two cases, the exit of mitosis would constitute a major and new import mechanism of proteins into the nucleus, possibly linking nucleus and cytoplasm in a way previously not appreciated. This might play a role, for example in linking transcription to metabolism, or simply being evolution “at work”, creating opportunities for new functions by creating protein proximities and environments. We hope to offer you some answers in our next Centre brochure.

3D proteomics and chromatin proteomics

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Selected Publications: Chen, Z.A., Jawhari, A., Fischer, L., Buchen, C., Tahir, S., Kamenski, T., Rasmussen, M., Lariviere, L., Bukowski-Wills, J.C., Nilges, M., Cramer P, and Rappsilber J. (2010). Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J 29, 717-726.

Rappsilber, J. (2011). The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes. J Struct Biol 173, 530-540.

Ohta, S., Bukowski-Wills, J.C., Sanchez-Pulido, L., Alves Fde, L., Wood, L., Chen, Z.A., Platani, M., Fischer, L., Hudson, D.F., Ponting, C.P., Fukagawa T., Earnshaw* W.C., and Rappsilber* J. (2010). The protein composition of mitotic chromosomes determined using multi-classifier combinatorial proteomics. Cell 142, 810-821.

Figure 1. New web tools for intuitive data interrogation and hypothesis generation based on mass spectrometry data of cross-linked peptides. The web-based graphical user interface of our in-house database search software Xi, now comprises highly interactive spectrum and protein network viewers that are based on the latest web technology. Scientists can interrogate spectra-peptide matches and linkage networks with minimal requirements for expert knowledge. This forms the basis for a roll out of our tools to the wider biology community.

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Kenneth E. Sawin

Co-workers: Weronika Borek, Eric Lynch, Hilary Snaith, Calum Stevenson.

We study the eukaryotic microtubule cytoskeleton, using fission yeast Schizosaccharomyces pombe as a model system, and combining classical and molecular genetics with microscopy and biochemistry/proteomics. Our goals are to understand how the cytoskeleton is organized, and how it functions to regulate cell-polarity landmarks.

Microtubule nucleation depends on the γ-tubulin complex (γ-TuC), a multi-protein complex enriched at microtubule organizing centers such as the centrosome. Differentiated cells often have multiple, diverse microtubule organizing centers. The mechanisms that target the γ-tubulin complex to different sites are unknown, as are the mechanisms that activate the γ-tubulin complex. The fission yeast proteins Mto1 and Mto2 form an oligomeric complex (Mto1/2 complex) that binds the γ-TuC and targets it to different sites during the cell cycle. Mutations in the human homolog of Mto1 lead to the brain disease microcephaly. Our recent work indicates that Mto1/2 not only localizes the γ-TuC but also activates it, possibly by inducing conformational changes upon binding. In addition, the molecular architecture of the Mto1/2 complex is regulated by phosphorylation during the cell cycle. Current work is focused on cell-cycle regulation, as well as the detailed biochemistry and structure of the Mto1/2 complex, for which we are expressing and purifying recombinant protein complexes in insect cells.

One important biological function of microtubules is to direct positioning of cell-polarity factors. In fission

yeast, the protein Tea1 associates with growing ends of microtubules until they reach the cell tips, whereupon Tea1 is deposited at the cell cortex. Using a combination of genetics, FRAP microscopy, and mathematical modelling we identified a novel mechanism in which the membrane protein Mod5 plays a catalytic role in promoting cortical anchoring of Tea1. Our recent studies suggest that Tea1 turnover at cell tips may be regulated by cumulative phosphorylation. In a separate project identifying novel Tea1-interacting proteins, we have unexpectedly found that proteins associating with Tea1 contribute to exocytosis.

An important element of our research involves making new tools for fission yeast genetics, microscopy, biochemistry and proteomics. Recently we developed a robust platform for differential proteomics in fission yeast, using Stable Isotope Labeling by Amino Acids in Culture (SILAC), as well as a novel matrix for rapid large-scale purification of tagged proteins from yeast.

Selected Publications: Anders, A., and Sawin, K.E. (2011). Microtubule stabilization in vivo by nucleation-incompetent {gamma}-tubulin complex. J Cell Sci 124, 1207-1213.

Bicho, C.C., Kelly, D.A., Snaith, H.A., Goryachev, A.B., and Sawin, K.E. (2010). A catalytic role for Mod5 in the formation of the Tea1 cell polarity landmark. Curr Biol 20, 1752-1757.

Samejima, I., Miller, V.J., Rincon, S.A., and Sawin, K.E. (2010). Fission yeast Mto1 regulates diversity of cytoplasmic microtubule organizing centers. Curr Biol 20, 1959-1965.

Microtubule nucleation and cytoskeletal organization

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Figure 1. Cell-cycle regulation of the Mto1/2 complex by phosphorylation of Mto2. Western blot showing Mto2 and Mto2 with 17 serine/threonine residues mutated to alanine, in interphase and mitosis. Mitotic phosphorylation of Mto2 disrupts its interaction with Mto1.

Int IntMit Mit

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Figure 2. Microtubule dynamics in an mto1-domain-swap mutant. The gamma-tubulin complex-binding region of Mto1 was replaced with a comparable region from the human protein myomegalin. In spite of limited sequence similarity, the human sequence is functional in fission yeast, indicating that structural interactions are conserved. Microtubules are in green, and Mto1/2 complex is in red.

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Eric C. Schirmer

Co-workers: Dzmitry G. Batrakou, Hannah Elam, Jose de las Heras, Vassiliki Lazou,Alexandr Makarov, Michael I Robson, Vlastimil Srsen, Nikolaj Zuleger.

Mutations in ubiquitous nuclear envelope (NE) proteins cause a wide range of diseases with tissue-specific pathologies from muscular dystrophies to lipodystrophies, neuropathy, osteopoikilosis, and premature aging syndromes. To explain how mutations in ubiquitous proteins can yield tissue-specific pathologies we postulated that as yet unidentified tissue-specific partners mediate the pathologies. In search of such proteins we have determined the proteome of NEs isolated from liver, muscle and two activation states of leukocytes. Interestingly, the majority of NE transmembrane proteins (NETs) identified were tissue-specific. Bioinformatic analysis of the previously characterised proteins identified suggests that this tissue-specific complexity supports tissue-specific regulation of signaling and gene expression and thus could explain the many diseases now linked to the NE. We have cloned roughly 80 uncharacterized tissue-specific NETs and used them in screens to identify those that influence cytoskeletal organisation, cell cycle progression, differentiation and genome organization.

Of particular note, several tissue-specific NETs affect the positioning of certain chromosomes with respect to the nuclear periphery. Their expression in cell types where they are not normally expressed resulted in these chromosomes repositioning from the nuclear interior to the nuclear periphery while their knockdown in cells where they are normally expressed resulted in the relocation of certain chromosomes away from the nuclear periphery.

For example, chromosome 5 is normally in the nuclear interior in fibroblast cells and at the nuclear periphery in liver. Exogenous expression of liver-specific NET47 in a general fibroblast line recruited chromosome 5 to the periphery while its knockdown in liver cells caused chromosome 5 to move away from the periphery. The change in chromosome position was accompanied by changes in gene expression. NET47 expression in the fibroblasts induced several genes that are normally upregulated in liver differentiation. Other NETs that were preferentially expressed in muscle cells or adipocytes had the same types of effects and could thus contribute to muscular dystrophies and lipodystrophies linked to the NE and potentially also to diabetes and obesity. We are currently investigating the correlation between specific gene repositioning and changs in expression using various differentiation systems.

Separately, we are working to determine the interaction partners of NETs that influence cytoskeletal organisation and investigating their roles in establishment of tissue-specific cell polarity, studying herpesvirus interactions with the NE which plays a crucial yet ill defined role in the virus life cycle, and performing exome sequencing in unlinked muscular dystrophy patients to identify additional disease variants.

Nuclear envelope transmembrane protein regulation of tissue-specific genome and cytoskeletal organization

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Selected Publications: Malik, P., Tabarraei, A., Kehlenbach, R. H., Korfali, N., Iwasawa, R., Graham, S. V., and Schirmer, E. C. (2012) Herpes simplex virus ICP27 protein directly interacts with the nuclear pore complex through Nup62, inhibiting host nucleocytoplasmic transport pathways. J. Biol. Chem. 287(15), 12277-12292.

Zuleger, N., Kelly, D. A., Richardson, A. C., Kerr, A. R. W., Goldberg, M. W., Goryachev, A. B., and Schirmer, E. C. (2011) System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics. J. Cell Biol. 193, 109-123.

Wilkie, G. S., Korfali, N., Swanson, S. K., Malik, P., Srsen, V., Batrakou, D. G., de las Heras, J., Zuleger, N., Kerr, A. R. W., Florens, L., and Schirmer, E. C. (2011) Several novel nuclear envelope proteins from muscle have cytoskeletal associations. Mol. Cell. Proteomics 10, M110.003129.

A. The nuclear envelope proteome varies between tissues. Proteins identified in blood, muscle or liver were plotted by their relative expression in the three tissues according to the BioGPS transcriptome database. They were then color coded according to the tissue where they were identified: blood (red), muscle (yellow), liver (blue), blood and muscle (orange), blood and liver (purple), muscle and liver (green), or all three tissues (brown). Correlation between transcriptome and proteomics results confirms the nuclear envelope tissue variation. B. NETs that alter chromosome positioning. Chromosome 5 is normally in the nuclear interior and most NETs (NET37 shown) do not affect its positioning. However, NET29, NET39, and NET47 all increased its incidence at the nuclear periphery. C. In addition to repositioning chromosomes, liver-specific NET47 upregulates liver-specific gene expression. Genes upregulated by NET47 expression in HT1080 fibroblast cells are plotted according to their degree of upregulation in a study of hepatic differentiation. Positions above the diagonal are associated with upregulation in the hepatic study. The genes upregulated due to NET47 expression are colored red and those downregulated are colored blue. Most NETs upregulated by NET47 occur above the diagonal, indicating that NET47 upregulates genes that are also upregulated in hepatic differentiation. D. Some NETs interact with the microtubule spindle during mitosis. Most NETs have no particular association with the mitotic spindle (e.g. SUN2 and Rhbdd1). In contrast, Tmem214 tracks with microtubules in the assembling mitotic spindle in prophase. WFS1 has a different distribution, accumulating preferentially around the base of the spindle poles during mitosis.

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Irina Stancheva

Co-workers: Joe Burrage, Katrina Gordon, Sadie Kemp, Chao Li, Ausma Termanis, Tuo Zhang.

Epigenetic Gene Silencing in Development and Cancer

We study how DNA methylation and silencing histone modifications contribute to regulation of gene expression in mammalian cells. In particular, we investigate the molecular mechanisms that guide the establishment of DNA methylation patterns in embryogenesis and lineage commitment, as well as during cellular transformation into cancer. We also study the crosstalk between DNA methylation and histone modifications during the establishment and propagation of silenced chromatin states.

In early mouse development, lineage-specific DNA methylation patterns are established in the post-implantation embryo by DNA methyltransferase enzymes DNMT3A and DNMT3B. However, there are additional proteins that facilitate the action of DNMT enzymes either globally or in a locus-specific manner. We recently found that, in the absence of chromatin remodelling ATPase LSH/Hells DNA methylation patterns at specific loci cannot be correctly established in embryonic lineage cells leading to missexpression of a large number of genes (Myant et al, 2011). Interestingly, wild-type LSH, but not catalytically inactive LSH (Figure 1A), can partly restore DNA methylation in Lsh-/- mouse embryonic fibroblasts at repetitive sequences and gene promoters (Figure 1B-C) resulting in re-silencing of associated genes. These data demonstrate that LSH can promote de novo DNA methylation in a cell- autonomous manner and further suggest that chromatin remodelling by LSH facilitates the processivity of its interacting partner DNMT3B (Figure 1D;

Myant and Stancheva 2008). Independently of its role in DNA methylation, LSH is required for efficient repair of DNA double-stand breaks (DSBs) in somatic cells. We are currently using mouse models, genomic, proteomic and biochemical approaches in order to unravel the mechanistic details of how LSH remodels chromatin to promote DNA methylation and DNA repair. We also study how other regulators of DNA methylation, in particular G9a/GLP complex of histone methylases, facilitate establishment and maintenance of DNA methylation in mammalian cells.

The conversion of normal cells into cancer typically involves several steps resulting in the acquisition of unlimited growth potential (immortality). Both genetic and epigenetic changes have been detected in a number of different cancer cell types. Although a number of tumour suppressor genes can be silenced by promoter DNA methylation, it is yet unclear whether epigenetic changes contribute directly to cancer and if so when, where and how do they arise. In order to address these questions and study the global epigenetic landscape associated with cellular immortalisation and transformation, we generated a human cancer cell model with defined genetic characteristics. Following the epigenetic changes in these cells, we found accumulation of DNA methylation at specific promoters and upregulation of genes promoting cell survival. These changes take place at specific time, but are largely independent of introduced oncogenes.

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Figure1. Chromatin remodelling ATPase LSH can restore DNA methylation in Lsh-/- mouse embryonic fibroblasts (MEFs) A. Predicted structure of chromatin remodelling domain of LSH protein. ATP binding site and the point mutation K237Q that abolish ATP binding are indicated. B. Quantification of DNA methylation in wild-type MEFs, Lsh-/- MEFs and clonal cell lines derived from Lsh-/- MEFs expressing either wild-type or catalytically inactive LSH protein. C. Southern blot of DNA from indicated cell lines digested with methylation-insensitive MspI or methylation-sensitive HpaII restriction enzyme detects DNA methylation at mouse centromeric sequences. D. Chromatin remodelling by LSH facilitates processivity of DNMT3B.

Selected Publications: Myant, K. and Stancheva, I. (2008) LSH cooperates with DNA methyltransferases to repress transcription., Mol Cell Biol, 28, 215-226.

Myant, K., Termanis, A., Sundaram, A. Y., Boe, T., Li, C., Merusi, C., Burrage, J., de Las Heras, J. I., and Stancheva, I. (2011). LSH and

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G9a/GLP complex are required for developmentally programmed DNA methylation. Genome Res 21, 83-94.

Stancheva, I. (2011) Revisiting heterochromatin in embryonic stem cells, PLoS Genetics 7(6), e1002093.

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David Tollervey

Co-workers: Konstantine Axt, Clémentine Delan-Forino, Aziz El Hage, Tatiana Dudnakova, Aleksandra Helwak, Rebecca Holmes, Grzegorz Kudla, Simon Lebaron, Laura Milligan, Elisabeth Petfalski, Alex Tuck.

Nuclear RNA Processing and Surveillance

Our aim is to understand the nuclear pathways that process newly transcribed RNAs and assemble the RNA-protein complexes, the mechanisms that regulate these pathways and the surveillance activities that monitor their fidelity. We are addressing these questions using a combination of systems biology, genetics, biochemistry and cell biology approaches in the budding yeast Saccharomyces cerevisiae.

To better understand both RNA maturation and surveillance, we developed techniques for quantitative analysis supported by mathematical modelling (Kos and Tollervey, 2010) and for UV cross-linking and analysis of cDNAs (CRAC) to precisely identify sites of RNA-protein interaction (Granneman et al., 2009). These are having a dramatic effect on our understanding of ribosome synthesis (Granneman et al., 2011; van Nues et al., 2011; Lebaron et al., 2012). Analyses of factors required for the final steps in 18S rRNA maturation revealed unexpected roles for the translation initiation factor eIF5b and mature 60S subunits in the establishment of pre-rRNA cleavage competence in late pre-40S particles (Lebaron et al., 2012). As outlined in Fig. 1, eIF5b associates with the pre-40S particles in association with mature 60S particles. The GTPase activity of eIF5b is then predicted to displace the pre-rRNA spacer region and drive rotation of the head region of the small subunit, bringing the active site of the Nob1 endonuclease into proximity with the cleavage site at the 3’ end of the 18S rRNA. The requirement for

interactions between late pre-40S particles and both 60S subunits and a translation factor is likely to constitute a proof reading step that assesses subunit functionality prior to final activation for translation. The factors involved are highly conserved and we predict that this is an ancient quality control system.

The identification of RNA-RNA interactions is also essential for the detailed understanding of many biological processes. We have described a high-throughput method to experimentally identify intramolecular and intermolecular RNA-RNA interactions by crosslinking, ligation and sequencing of hybrids (CLASH) (Kudla et al., 2011). Application of this approach to human Argonaut protein 1 (Ago1) has identified 18,000 high confidence miRNA-mRNA interaction sites, and transformed miRNA target prediction from a complex bioinformatics problem to an experimental technique. In addition to mRNAs, we found numerous miRNA-binding sites on diverse RNAs, including rRNAs, tRNAs and other miRNAs, and we speculate these interactions help regulate miRNA activity. Our discoveries give important insights into miRNA targeting patterns and the miRNA interactome.

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Selected Publications: Kudla, G., Granneman, S., Hahn, D., Beggs, J.D. and David Tollervey, D. (2011) Crosslinking, ligation and sequencing of hybrids reveals RNA-RNA interactions in yeast. Proc. Natl. Acad. Sci. USA. 108, 10010-10015.

Granneman, S., Petfalski. E. and Tollervey, D. (2011) A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease. EMBO J. 30, 4006-4019.

Lebaron, S., Schneider, C., van Nues, R.W., Swiatkowska, A., Walsh, D., Böttcher, B,. Granneman, S., Watkins, N.J. and Tollervey, D. (2012) Proof reading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nat. Struct. Mol. Biol. Online publication July 1, 2012.

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Figure 1. Model of steps driving 3’ cleavage of the 18S rRNA. A. The pre-rRNA spacer is proposed to be located in the mRNA-binding cleft. B. eIF5b (purple) binds pre-40S particles together with the 60S subunit. C. GTP hydrolysis by eIF5b drives movement of the head domain and displaces the pre-rRNA within the mRNA binding cleft, bringing the cleavage site together with the endonuclease Nob1. D. rRNA cleavage and release of Nob1 generates mature, translation-competent 40S subunits.

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Mike Tyers

Co-workers: Emilie Castonguay, Aileen Greig, Joanna Koszela, Vassiliki Lazou,Eléonore Lebaron, Julie Nixon, Lindsay Ramage, Marcia Roy, Michaela Spitzer, Hille Tekotte, Kim Webb, Rachel White, Jan Wildenhain, Andrew Winter.

All cells must coordinate cell growth with division, usually before commitment to division in late G1 phase. Our systematic phenotypic and genetic screens in budding yeast have revealed hundreds of factors that influence cell size, including an Rb-like inhibitor of G1/S transcription called Whi5, a transcription factor called Sfp1 that governs expression of ribosome biogenesis genes, an S6 kinase equivalent called Sch9 that lies downstream of the TORC1 nutrient sensing complex, and the Rab escort protein Mrs6 that connects vesicle trafficking with ribosome biogenesis and cell size.

We have recently established a new link between Whi5 and mitochondrial function. In a screen for new cell size regulators that interact with Whi5, we identified 68 yeast gene deletions that are inviable when Whi5 is over-expressed, 56% of which have known mitochondrial functions (Fig. 1A). One of these genes encodes Ptc1, a type 2C phosphatase that is required for proper mitochondrial inheritance. Reduced or excessive Whi5 dosage perturbs mitochondrial morphology and inheritance in the absence of Ptc1 (Fig. 1B). We speculate that Whi5 may link mitochondrial replication and segregation to the cell-division cycle, and that mitochondrial function influences cell size. Our genetic data further suggest that Sch9 may relay mitochondrial activity to the size control machinery.

Our analysis of the protein interactions of all kinases and phosphatases in yeast by mass spectrometry revealed a host of new interactions for the TOR nutrient sensing network.

One of the novel TORC1 interactors is a kinase called Nnk1, which itself forms a stoichiometric complex with the metabolic enzyme Gdh2 that catalyzes the deamination of glutamate to α-ketoglutarate. Nnk1 appears to control the localization and activity of Gdh2 (Fig. 1C), and thereby may link TOR activity to carbon and nitrogen metabolism. Our genetic screens for modulators of Gdh2 localization have revealed a number of other interconnections with known nutrient sensing networks.

We have undertaken genome-wide approaches to chart chemical genetic space, with the goal of rationally controlling cellular function with combinations of small molecules. Our screens for synergistic combinations of molecules have revealed potential new anti-infective agents, while screens with focused libraries of bioactive compounds against budding yeast, fission yeast, nematodes, worms, zebrafish, thale cress and mammalian cell lines have uncovered many new chemical probes for biological function. Additional screens are underway for molecules that influence epigenetic silencing of heterochromatin and for inhibitors of E2 enzymes in the ubiquitin-proteasome system. These datasets have been integrated through a custom database and an associated set of chemoinformatic tools.

Finally, we have continued to expand our open access protein and genetic interaction database called the BioGRID (www.thebiogrid.org), which is populated through international curation efforts in the UK, US and Canada. We and others use these BioGRID datasets as a resource to understand gene function and to interrogate the structure of cellular networks.

Systems biology of cell growth and division

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Selected Publications: Tang X, Orlicky S, Mittag T, Csizmok V, Pawson T, Forman-Kay JD, Sicheri F and Tyers M. (2012) Composite low affinity interactions set a multisite phosphorylation threshold for recognition of Sic1 by the SCFCdc4 ubiquitin ligase. Proc Natl Acad Sci USA 109:3287-3292.

Ceccarelli DF, Tang X, Pelletier B, Orlicky S, Neculai D, Chou Y-C, Ogunjimi A, Al-Hakim A, Varelas X, Koszela J, Wasney G, Vedadi M, Dhe-Paganon S, Xie W, Plantevin V, Cox S, Lopez-Girona A,

Figure 1A. Network of mitochondrial and cell size genes that exhibit synthetic lethality with WHI5 overexpression. B. Severe defect in mitochondrial inheritance of a whi5∆ ptc1∆ strain revealed by real time microfluidic analysis. Circles show daughter buds that fail to inherit mitochondria. C. Nnk1-dependent Gdh2-GFP focus formation upon nutrient limitation.

Mercurio F, Wrana J, Durocher D, Meloche S, Webb DR*, Tyers M*, Sicheri F* (2011) An allosteric inhibitor of the human Cdc34 ubiquitin conjugating enzyme. Cell 145, 1075-1087.

Spitzer M†, Griffiths E†, Blakely KM, Wildenhain J, Ejim J, Rossi L, De Pascale G, Curak J, Brown E, Tyers M*, Wright GD* (2011) Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole. Mol Sys Biol 7, 499.

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Malcolm Walkinshaw

Co-workers: Elizabeth Blackburn, Janice Bramham, Sandra Bruce, Yiyuan Chen, Jacqueline Dornan, Douglas Houston, Iain McNae, Neil McKenzie, Hugh Morgan, Ankita Naithani, Matt Nowicki, Rosie Palmer, Calum Stevenson, Paul Taylor, Montserrat Vasquez, Martin Wear, Li Hsuan Yen, Wenhe Zhong.

We are interested in the molecular mechanisms that communicate information in signal transduction and metabolic pathways. Small molecule effector or inhibitor molecules are frequently used to control protein shape or inhibit enzyme activity. In the glycolytic pathway for example a series of 10 enzymes break down glucose to give pyruvate and each of the ten chemical stages is carefully regulated by numerous allosteric and feedback mechanisms. We have studied this glycolytic pathway in trypanosomatid parasites in order to identify allosteric mechanisms that could lead to species specific inhibitors and provide drug-leads against diseases including sleeping sickness, leishmaniasis and Chaga’s disease.

We have now captured X-ray structures of a range of allosteric states of trypanosomatid phosphofructokinase (PFK) and pyruvatekinase (PYK). These structures provide details of their allosteric control mechanisms (i.e. how does the presence of one ligand affect the binding of another ligand at a geographically remote site). A series of X-ray structures of PFK and PYK complexed with different inhibitors, effectors and substrates explain how allosteric ligands binding remotely from the enzyme active sites can have such a profound effect on the enzyme activity.

A parallel study of the X-ray and biochemical properties of human isoforms of PYK show interesting examples of divergent molecular evolution in which the catalytic mechanism is strictly conserved at a molecular level, however the allosteric mechanisms that regulate activity

have evolved in a variety of very different ways. The human M2PYK isoform is up-regulated in almost all tumour cells and we have shown that enzyme activity is governed by the equilibrium between (inactive) monomers and (active) tetramers. The monomer-tetramer balance is regulated by both protein concentration and small molecule effectors and inhibitors (Figure). By screening against a small library of metabolite molecules we have also identified a number of metabolites that allosterically inhibit M2PYK by trapping the tetramer in an inactive conformation. Structurally this is achieved by a tryptophan flip that locks the tetramer in an inactive conformation. The LmPYK enzyme has evolved a different mechanism to stabilise its active and inactive states and uses a set of salt bridges to lock the tetramer in an active conformation. A second class of M2PYK inhibitors is exemplified by the thyroid hormone triiodo-thyronine (T3) which binds tightly (Kd = 80 nM) to monomers of M2PYK keeping the enzyme catalytically inactive.

We have now made a link between small molecule activation and inhibition of M2PYK and their effect on cell proliferation (Figure). For cancer cell lines that over express M2PYK, inhibitors of M2PYK enzyme activity dramatically increased cell proliferation while the activator F16BP reduced proliferation. The working hypothesis is that blocking the final step in glycolysis causes a build-up of intermediate metabolites which are required for synthesis of protein and DNA in growing and dividing cells.

Molecular Recognition in Biological Systems

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Selected Publications: Husi, H., McAllister, F., Angelopoulos, N., Butler, V. J., Bailey, K. R., Malone, K., Mackay, L., Taylor, P., Page, A. P., Turner, N. J., et al. (2010). Selective chemical intervention in the proteome of Caenorhabditis elegans. J Proteome Res 9, 6060-6070.

Morgan, H. P., McNae, I. W., Nowicki, M. W., Hannaert, V., Michels, P. A., Fothergill-Gilmore, L. A., and Walkinshaw, M. D. (2010b).

Allosteric mechanism of pyruvate kinase from Leishmania mexicana uses a rock and lock model. J Biol Chem 285, 12892-12898.

Richardson, J. M., Colloms, S. D., Finnegan, D. J., and Walkinshaw, M. D. (2009a). Molecular architecture of the Mos1 paired-end complex: the structural basis of DNA transposition in a eukaryote. Cell 138, 1096-1108.

Figure 1. Allosteric regulation of M2pyruvate kinase activity regulates cell proliferation. X-ray structures of the tetrameric Phe-bound T-state and F16BP-activeted R-state are shown as cartoons a,b,c,d) with each 50Kda protomer represented by a rectangular shape showing the allosteric sites. M2PYK exists in a concentration dependent equilibrium between tetrameric and enzymatically inactive monomer forms with a Kd ~8µM. Phenylalanine (Phe, green square) and the thyroid hormone triiodothyronine (T3, orange) act as allosteric inhibitors and prevent the tetramer adopting an active R-state conformation. The activator fructose 16 bisphosphate (F16BB) clamps the tetramer in an enzymatically active conformation. Addition of F16BP to HCT-116 cells cells inhibits proliferation of while both inhibitors of M2PYK (T3 and Phe) cause cell proliferation (e).

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Julie WelburnCo-worker: Sarah Young

Regulation of microtubules in mitosis

Mitosis is a highly regulated process that allows equal distribution of the genetic material to the daughter cells. The mitotic spindle is a bipolar structure composed of highly dynamic microtubule polymers. The formation and maintenance of the bipolar spindle, kinetochore-microtubule attachments, chromosome movement and microtubule dynamics during mitosis are critical to maintaining genomic integrity. Several of the mitotic microtubule-associated proteins are key to controlling these processes and therefore prime candidates as anti-cancer drug targets.

Our goals are to determine how the key mitotic players that contribute to correct chromosome segregation are controlled, and the molecular principles that govern the progression of mitosis. Aneuploidy and chromosome instability can arise from defects in chromosome segregation and are a hallmark of cancer. Therefore, mitotic molecular motors and other microtubule-associated proteins that control spindle assembly and chromosome segregation represent key cancer drug targets to be further evaluated. Using biochemistry and structural biology, we study the architecture of cell cycle complexes, while cell biology approaches give us a cellular context to understand how molecular machines work.

Several of the mitotic microtubule-associated proteins termed kinesins are key to controlling these processes and therefore prime candidates as anti-cancer drug targets. While the majority of kinesin proteins are microtubule-

based motors, the kinesin-13 family members are microtubule depolymerizing enzymes that are major mechanistic players in spindle assembly and chromosome movement.

We are focusing on the 3 members of the essential kinesin-13 family. Despite a highly conserved motor domain, kinesin-13s display different localization during mitosis and divergent functions in spindle assembly, correction of erroneous kinetochore-microtubule attachments, and chromosome movement. We have determined intrinsic factors, such as kinesin domains, and extrinsic factors, such as associated proteins, that create kinesin specificity to regulate the function and localization of the kinesin-13 proteins. We have showed that these factors control specifically and uniquely the targeting to kinetochores, centrosomes and microtubules of kinesins. In particular, we have identified the spindle-localized proteins Cep170 and Cep170R (KIAA0284), as specifically associating with Kif2b. Cep170 binds to microtubules in vitro and provides the Kif2b holo-complex with a second microtubule binding site to target it to the spindle. Thus, the intrinsic properties of kinesin-13s and extrinsic factors such as their associated proteins result in the diversity and specificity within the kinesin-13 depolymerase family.

Future aims will include defining the architecture and dissect the biochemical properties of kinesin-13 complexes.

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Selected Publication: Welburn, J.P., Vleugel, M., Liu, D., Yates, J.R., 3rd, Lampson, M.A., Fukagawa, T., and Cheeseman, I.M. (2010). Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface. Mol Cell 38, 383-392.

A. Images of human HeLa cells expressing GFP fusions to full length and domains of Kif2a, Kif2b and Kif2c. The ∆N and ∆MC domains describe GFP-fusions lacking the N-terminus and the motor and C-terminal domains of kinesins respectively. B. Co-sedimentation of Cep170 at the indicated microtubule concentrations showing that Cep170 binds to microtubules. C. Images of live mitotic cells expressing mCherry-Kif2b under control conditions or following Cep170 or Cep170R RNAi-depletion, showing that Kif2b targeting to the spindle requires Cep170. D. Schematic diagram showing how Kif2b associates with Cep170 to target to the spindle. The complex has 2 binding sites, that consequently enhances the affinity of Kif2b-Cep170 for microtubules.

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Steve West

Co-workers: Lee Davidson, Anne Hett, Lisa Muniz.

Mechanisms of nuclear pre-mRNA surveillance in human cells

Most cells in the human body have the same DNA content, yet not all cells are the same. This is because different cells have different gene expression programmes. It is the specific genes that are expressed and the level of their expression that determines cell phenotype and whether the cell is healthy or not. One of the most relevant classes of genes code for proteins and there are 20-25 thousand within the human genome. Their expression is achieved by transcription of the DNA into pre-messenger RNA (pre-mRNA), which is matured by capping, splicing and 3’ end processing. The complete mRNA is then exported from the nucleus to the cytoplasm where it is translated into protein. Maintaining accuracy during gene expression is very important and is achieved by a number of RNA quality control mechanisms that lead to degradation of transcripts that are improperly synthesised or processed.

My lab is focussed on characterising mechanisms of RNA quality control that operate on protein-coding transcripts in the human nucleus. We are particularly interested in quality control of splicing and 3’ end processing of pre-mRNA. We are keen to work out how and when improperly processed pre-mRNAs are recognised for degradation and identify the enzymes that execute their decay. A range of in vivo and in vitro RNA labelling techniques are utilised to determine the stability of different pre-mRNA transcripts and to quantitate changes induced by cis- mutation or depletion of exoribonucleases and RNA processing factors. Our recent findings implicate several nuclear exoribonuclease

enzymes in the degradation of improperly processed pre-mRNA. We find that many defective pre-mRNAs are co-transcriptionally degraded implying that there are frequently very rapid means of identifying aberrant RNA.

Current research aims to characterise the fate of pre-mRNAs whose synthesis is blocked at defined stages during maturation. These studies are aimed at identifying different pathways of pre-mRNA turnover and the proteins implicated in them.

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Selected publications: Davidson L, Kerr A and West S (2012). Co-transcriptional degradation of aberrant pre-mRNA by Xrn2. EMBO Journal. April 20;31(11): 2566-78.

West S and Proudfoot NJ. (2009). Transcriptional termination enhances protein expression in human cells. Mol Cell. Feb 13;33(3):354-64.

West S, Gromak N, Norbury CJ, Proudfoot NJ. (2006). Adenylation and exosome-mediated degradation of cotranscriptionally cleaved pre-messenger RNA in human cells. Mol Cell. Feb 3;21(3):437-43.

West S, Gromak N, Proudfoot NJ. (2004). Human 5’ --> 3’ exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature. Nov 25;432(7016):522-5.

Figure 1. The 5→3’ exonuclease, Xrn2, degrades many aberrant pre-mRNAs following inhibition of splicing.Hela cells were treated with control or Xrn2 specific siRNAs and subsequently with or without spliceostatin A (SSA), which is a known splicing inhibitor. Nascent transcripts associated with chromatin were then analysed by quantitative RT-PCR. Knock-down of Xrn2 in the absence of SSA had no effect of RNA abundance (white versus grey bars). However, 5/6 transcripts were found in higher abundance upon depletion of Xrn2 when SSA was used (black versus red bars). The fold increase in RNA abundance for each transcript is displayed under the graph. This shows that Xrn2 degrades many pre-mRNAs when splicing is inhibited but not under normal circumstances.

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The Wellcome Trust Centre for Cell Biology is committed to engaging the public in science and our research. We carry out a host of activities to engage with school children, teachers, visitors to festivals and the general public.

Each year we are involved in and run a number of projects such as:

1. Life Through a Lens: Touring schools with our workshop/drama

2. Drop in days for the general public: at the Botanic Gardens

3. Edinburgh International Science Festival

4. Parent’s Like Us Festival: our centre runs the science tent

5. Orkney Science Festival: our first visit 2012

Some scientists within the centre also run their own outreach activities for example: Kick Start (labs with teenagers) and SSERC teachers training.

Life Through a Lens. This is a workshop & drama aimed at primary school pupils, and targeting schools in rural and deprived areas. It can be hard to get cell biology across to young children, so we decided to use acting and story telling to bring the subject alive. The response was tremendous: in November 2011 we had 16 slots for schools, and 45 schools applied.

In the play, the pupils meet four scientists from history and each one has a theory. The children demand that the scientist prove their theory. The scientists then help the children to perform their own experiments to prove or disprove the theory. The play ends with a modern day scientist from our centre talking about their current research. This workshop requires about 7 scientists to

run it and often scientists from our centre fill these roles, working with the pupils and also, if they are willing, as actors, playing the roles of historical scientists.

Parent Like Us festival. This year we ran the science tent with great success we had over 3000 visitors to our tent over two days. This festival, run by local residents, aims to reach young families with children under 6 years, with education based activities and to encourage learning through play.

Art. In 2011, I worked with three research scientists to produce etchings that represented their research and the history of cell biology, these were exhibited in the Royal Botanic Gardens in November 2011. In 2012, I have developed a pilot project using fused glass to illustrate concepts in modern cell biology and hope to work with several labs over the next 12 months using this technique.

Recent events: June 2012 Liberton High School. We trained senior pupils at this local high school to run our Life Through a Lens workshops. We then helped them deliver the workshop to six classes of primary school children visiting the High school.

June 2012 Leith. We ran the science tent at the Parents Like Us Festival in Leith, Edinburgh. We worked with over 3000 people over 2 days and had volunteers from many different labs at the WTCCB.

November 2011 Royal Botanical Gardens. Wellcome Trust 75th Anniversary celebration project. We ran four weekend drop-in days for the general public, with over 800 visitors. We also ran 16 sessions with schools, targeting rural and deprived areas with our travel bursary scheme, we worked with over 450 school pupils in total.

Sarah Keer-Keer

Outreach and Public Engagement

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Figure 1A,B,C. show children attending school workshop production of Life Through a Lens. D,E,F. show visitors to public drop in days.

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List of Groups

Robin Allshire Wellcome Trust Principal Research Fellow

Tatsiana Auchynnikava Wellcome Research AssociatePauline Audergon Wellcome 4yr Graduate StudentHarald Berger Austrian FWF Research AssociateAlessia Buscaino Wellcome Research AssociateEmilie Castonguy* NSERC Graduate StudentSandra Catania Wellcome 4yr Graduate StudentGeorgina Hamilton Wellcome Research AssistantErwan Lejeune EC Marie Curie Intra-European Research AssociateMatthew Miell SULSA Graduate StudentAlison Pidoux Wellcome Research AssociateManu Shukla EMBO Research AssociateLakxmi Subramanian EC Marie Curie Research AssociateJitendra Thakur Visitor (EMBO Short Term Fellowship)Nick Toda Darwin Trust Graduate StudentSharon White Wellcome Research Associate[*joint with Tyers lab]

A. Jeyaprakash Arulanandam Wellcome Research Career Development Fellow

Bethan Medina Wellcome Postgraduate Research AssistantMaria Alba Abad Fernandaz Part-time Research Associate

Jean Beggs Royal Society Darwin Trust Professor

Ross Alexander CSBE Research AssociateDavid Barrass Wellcome Research AssociateKeerthi Chathoth EC Unicellsys Graduate StudentOlivier Cordin Wellcome Research AssociateVanessa Cristão Gulbenkian Graduate StudentAmit Gautam Darwin Trust Graduate StudentEve Hartswood Wellcome Research AssociateDaniela Hahn Darwin Trust Graduate StudentSteve Innocente Wellcome Research AssociateJane Reid Wellcome 4yr Graduate Student

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Adrian BirdBuchanan Professor of Genetics, Edinburgh University

Kyla Brown ECAT Graduate StudentHelene Cheval AMR Research AssociateJustyna Cholewa-Waclaw RSRT Research AssociateThomas Clouaire Wellcome Research AssociateDina De Sousa Wellcome Research AssistantRobert Ekiert RSRT Research Assistant Jacky Guy Wellcome Research AssociateMartha Koerner Erwin-Schrödinger Research AssociateSabine Lagger EMBO Research AssociateMatthew Lyst Wellcome Research AssociateCara Merusi RSRT Research AssistantGabriele Schweikert EMBO/Marie Curie Research AssociateJim Selfridge Wellcome Research AssociateChristine Struthers PA to Adrian Bird/Centre AdminElisabeth Wachter Wellcome 4yr Graduate student

Atlanta Cook MRC Career Development Fellow

Uma Jayachandran Wellcome Research AsssistantUrszula Wolkowicz Wellcome PhD Student

Bill Earnshaw Wellcome Trust Principal Research Fellow

Helena Barysz Darwin Trust Graduate Student Dan Booth Wellcome Research AssociateMar Carmena Wellcome Research AssociateFlorence Gohard Wellcome 4yr Graduate StudentAlexander Kagansky Wellcome Research AssociateNuno Martins Graduate StudentOscar Molina EMBO Research AssociateHiromi Ogawa Wellcome Research TechnicianShinya Ohta JSPS Research AssociateDiana Papini Graduate StudentMelpi Platani Wellcome Research AssociateItaru Samejima Wellcome Research AssociateKumiko Samejima Wellcome Research AssociatePaola Vagnarelli Wellcome Research AssociateGiulia Vargiu Graduate StudentLaura Wood Graduate Student

Kevin Hardwick University Reader

Karen May Wellcome Research AssociateKostas Paraskevopoulos Wellcome Research AssociateSjaak van der Sar Wellcome Research Assistant/ Graduate studentOnur Sen EBSS Graduate StudentAlicja Sochaj SULSA/SBS Graduate StudentIvan Yuan Graduate studentJudith Zich Wellcome Research Associate

Lea Harrington Professor of Telomere Biology

Larissa Christian Jennifer Dorrens Wellcome Research AssistantLaura Gardano Postdoctoral Research AssociateFiona Pryde Wellcome Research AssociateFabo Pucci Wellcome 4yr Graduate StudentElisa Wong CR-UK Graduate Student

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Adele Marston Wellcome Trust Senior Research Fellow

Julie Blyth SULSA/Wellcome Research AssistantColette Connor Graduate StudentEris Duro Sir Henry Wellcome Research AssociateJosefin Fernius Wellcome Research AssociateStefan Galander Wellcome 4yr Graduate StudentOlga Nerusheva Darwin Trust Graduate StudentClaudia Schaffner Wellcome Research AssociateKitty Verzijlbergen EMBO Research AssociateNadine Vincenten Wellcome 4yr Graduate Student

Gracjan Michlewski MRC Career Development Award Fellow

Nila Roy Choudhury MRC Research AssistantJakub Nowak Wellcome 4yr Graduate Student

Hiro Ohkura Wellcome Trust Senior Research Fellow

Sally Beard Wellcome Research AssociateManuel Breuer Wellcome Research AssociateSara Clohisey CR-UK Graduate StudentNatalie Colombié Wellcome Research AssociateFiona Cullen Wellcome Research AssociateAgata Gtuszek Wellcome 4yr Graduate StudentHeather Grey Wellcome Research AssistantKarolina Mateja SULSA Graduate StudentElvira Nikalayevich Darwin Trust Graduate StudentHeather Syred BBSRC Graduate student

Juri Rappsilber Wellcome Trust Senior Research Fellow

Adam Belsom Wellcome Research AssociateJimi-Carlo Bukowski-Wills Wellcome Research AssociateZhuo Angel Chen Wellcome Research AssociateColin Combe Wellcome Research AssociateFlavia de Lima Alves Wellcome Research AssociateCristina Furlan SULSA/BBSRC Graduate StudentLutz Fischer Wellcome Research AssistantGeorg Kustatscher Wellcome Research AssociatePaul Mason Wellcome Research AssistantLauri Peil Mobilitas Research AssociateMichaela Spitzer ERC Research AssociateSalman Tahir Wellcome Research AssistantSakharam Waghmare Wellcome Research Associate (ISSF)Karen Wills Wellcome Research AssociateJuan Zou Wellcome Proteomics Data Analysis Manager

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Ken Sawin Professor of Cell Biology

Weronika Borek CR-UK Graduate StudentEric Lynch Darwin Trust Graduate StudentHilary Snaith Wellcome Research AssociateCalum Stevenson Wellcome Research Assistant

Eric Schirmer Wellcome Trust Senior Research Fellow

Dzmitry Batrakou Darwin Trust Graduate StudentHannah Elam MRC Graduate studentJose de las Heras Wellcome Research AssociateVassiliki Lazou* Wellcome Research AssistantAlexander Makarov MRC Graduate StudentMichael I Robson Wellcome 4yr Graduate StudentVlastimil Srsen Wellcome Research AssociateNikolaj Zuleger Wellcome Research Assistant

Irina Stancheva CRUK Senior Research Fellow

Joe Burrage BBSRC Case Graduate StudentKatrina Gordon CRUK Research AssociateSadie Kemp CRUK Research AssistantChao Li CRUK Research AssociateAusma Termanis CRUK Graduate StudentTuo Zhang Darwin Trust Graduate Student

David TollerveyDirector Wellcome Trust Principal Research Fellow

Konstantine Axt EU Research AssociateClémentine Delan-Forino Wellcome Research AssociateAziz El Hage Wellcome Research AssociateTatiana Dudnakova Wellcome Research AssociateAleksandra Helwak Wellcome Research AssociateRebecca Holmes Sir Henry Wellcome Research FellowGrzegorz Kudla EMBO Research AssociateSimon Lebaron Wellcome Research AssociateLaura Milligan BBSRC Research AssociateElisabeth Petfalski Wellcome Research AssociateAlex Tuck Wellcome Graduate Student

Mike Tyers C H Waddington Chair of Systems Biology

Emilie Castonguay* NSERC Graduate StudentAileen Greig Wellcome Research AssistantJoanna Koszela Wellcome Graduate StudentVassiliki Lazou Eléonore Lebaron Wellcome Research AssociateJulie Nixon BBSRC Research AssociateLindsay Ramage Wellcome Research AssociateMarcia Roy Graduate StudentMichaela Spitzer** Wellcome Research AssociateHille Tekotte Wellcome Research AssociateKim Webb Wellcome Research AssociateRachel White ERC Research AssistantJan Wildenhain ERC Research AssistantAndrew Winter BBSRC Research Associate[*joint with Tyers lab] [**joint with Rappsibler lab]

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Malcolm Walkinshaw Chair of Structural Biochemistry

Elizabeth Blackburn SULSA BCF Research AssociateJanice Bramham SULSA BCF Facility ManagerSandra Bruce EPPF/Group Laboratory Manager Yiyuan Chen Graduate StudentJacqueline Dornan Research AssociateDouglas Houston University LecturerIain McNae Research AssociateNeil McKenzie BBSRC Graduate StudentHugh Morgan Research AssociateAnkita Naithani Darwin Trust Graduate StudentMatt Nowicki SULSA Research AssociateRosie Palmer Wellcome 4yr Graduate StudentCalum Stevenson BBSRC Case Graduate StudentPaul Taylor University Senior Lecturer/ Wellcome Research AssociateMontserrat Vasquez Graduate StudentMartin Wear EPPF Facility ManagerLi Hsuan Yen BBSRC Case Graduate StudentWenhe Zhong Graduate Student

Julie Welburn CR-UK Research Career Development Fellow

Sarah Young CR-UK Research Assistant

Steve West Wellcome Trust Career Development Fellow

Lee Davidson Wellcome Research AssistantAnne Hett Graduate StudentLisa Muniz EMBO Research Associate

Administration/Support Staff

Greg Anderson Centre Laboratory ManagerFlavia de Lima Alves Wellcome Research AssociateDavid Kelly Centre Microscopy Research ManagerColin McLaren Computing SupportAlastair Kerr BioinformaticianChristine Struthers PA to Adrian Bird/Centre AdminPaul Taylor Computing SupportKaren Traill Centre Manager/Wellcome 4yr PhD Programme AdministratorShaun Webb Bioinformatic supportJuan Zou Proteomics Data Analysis ManagerSarah Keer-Keer Public Engagement and Outreach Manager

Technical Support

Lloyd Mitchell

Washing-up/Media

Denise Affleck Andrew Kerr Margaret Martin Donna Pratt

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Centre Publications 2010 - 2012

Aitken, S., Alexander, R.D., and Beggs, J.D. (2011). Modelling reveals kinetic advantages of co-transcriptional splicing. PLoS Comput Biol 7, e1002215. PMID: 22022255

Aitken, S., Robert, M.C., Alexander, R.D., Goryanin, I., Bertrand, E., and Beggs, J.D. (2010). Processivity and coupling in messenger RNA transcription. PLoS One 5, e8845. PMID: 20126621

Alexander, R., and Beggs, J.D. (2010). Cross-talk in transcription, splicing and chromatin: who makes the first call? Biochem Soc Trans 38, 1251-1256. PMID: 20863294

Alexander, R.D., Barrass, J.D., Dichtl, B., Kos, M., Obtulowicz, T., Robert, M.C., Koper, M., Karkusiewicz, I., Mariconti, L., Tollervey, D., et al. (2010). RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3’-end processing in Saccharomyces cerevisiae. RNA 16, 2570-2580. PMID: 20974745

Alexander, R.D., Innocente, S.A., Barrass, J.D., and Beggs, J.D. (2010). Splicing-dependent RNA polymerase pausing in yeast. Mol Cell 40, 582-593. PMID: 21095588

Anders, A., and Sawin, K.E. (2011). Microtubule stabilization in vivo by nucleation-incompetent gamma-tubulin complex. J Cell Sci 124, 1207-1213. PMID: 21444751

Anderson, H.E., Kagansky, A., Wardle, J., Rappsilber, J., Allshire, R.C., and Whitehall, S.K. (2010). Silencing mediated by the Schizosaccharomyces pombe HIRA complex is dependent upon the Hpc2-like protein, Hip4. PloS one 5 e13488. PMID: 20976105

Athanasiadou, R., de Sousa, D., Myant, K., Merusi, C., Stancheva, I., and Bird, A. (2010). Targeting of de novo DNA methylation throughout the Oct-4 gene regulatory region in differentiating embryonic stem cells. PLoS One 5, e9937. PMID: 20376339

Bayne, E.H., White, S.A., Kagansky, A., Bijos, D.A., Sanchez-Pulido, L., Hoe, K.L., Kim, D.U., Park, H.O., Ponting, C.P., Rappsilber, J., et al. (2010). Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity. Cell 140, 666-677. PMID: 20211136

Bergmann, J.H., Jakubsche, J.N., Martins, N.M., Kagansky, A., Nakano, M., Kimura, H., Kelly, D.A., Turner, B.M., Masumoto, H., Larionov, V., et al. (2012). Epigenetic engineering: histone H3K9

acetylation is compatible with kinetochore structure and function. J Cell Sci 125, 411-421. PMID: 22331359

Bergmann, J.H., Rodriguez, M.G., Martins, N.M., Kimura, H., Kelly, D.A., Masumoto, H., Larionov, V., Jansen, L.E., and Earnshaw, W.C. (2011). Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. EMBO J 30, 328-340. PMID: 21157429

Bicho, C.C., de Lima Alves, F., Chen, Z.A., Rappsilber, J., and Sawin, K.E. (2010). A genetic-engineering solution to the “arginine-conversion problem” in SILAC. Mol Cell Proteomics 9, 1567-1577. PMID: 20460254

Bicho, C.C., Kelly, D.A., Snaith, H.A., Goryachev, A.B., and Sawin, K.E. (2010). A catalytic role for Mod5 in the formation of the Tea1 cell polarity landmark. Curr Biol 20, 1752-1757. PMID: 20850323

Bizzari, F., and Marston, A.L. (2011). Cdc55 coordinates spindle assembly and chromosome disjunction during meiosis. J Cell Biol 193, 1213-1228. PMID: 21690308

Blackburn, E.A., Maclean, J.K., Sherborne, B.S., and Walkinshaw, M.D. (2010). Estimating the affinity of protein-ligand complex from changes to the charge-state distribution of a protein in electrospray ionization mass spectrometry. Biochem Biophys Res Commun 403, 190-193. PMID: 21056549

Blackburn, E.A., and Walkinshaw, M.D. (2011). Targeting FKBP isoforms with small-molecule ligands. Curr Opin Pharmacol 11, 365-371. PMID: 21803654

Bohnsack, M.T., Tollervey, D., and Granneman, S. (2012). Identification of RNA Helicase Target Sites by UV Cross-Linking and Analysis of cDNA. Methods Enzymol 511, 275-288. PMID: 22713325

Bono, F., Cook, A.G., Grunwald, M., Ebert, J., and Conti, E. (2010). Nuclear import mechanism of the EJC component Mago-Y14 revealed by structural studies of importin 13. Mol Cell 37, 211-222. PMID: 20122403

Braun, N., Zacharias, M., Peschek, J., Kastenmuller, A., Zou, J., Hanzlik, M., Haslbeck, M., Rappsilber, J., Buchner, J., and Weinkauf, S. (2011). Multiple molecular architectures of the eye lens chaperone alphaB-crystallin elucidated by a triple hybrid approach. Proc Natl Acad Sci U S A 108, 20491-20496. PMID: 22143763

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Buscaino, A., Allshire, R., and Pidoux, A. (2010). Building centromeres: home sweet home or a nomadic existence? Curr Opin Genet Dev 20, 118-126. PMID: 20206496

Buscaino, A., White, S.A., Houston, D.R., Lejeune, E., Simmer, F., de Lima Alves, F., Diyora, P.T., Urano, T., Bayne, E.H., Rappsilber, J., et al. (2012). Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification. PLoS Genet 8, e1002499. PMID: 22319459

Buttrick, G.J., Meadows, J.C., Lancaster, T.C., Vanoosthuyse, V., Shepperd, L.A., Hoe, K.L., Kim, D.U., Park, H.O., Hardwick, K.G., and Millar, J.B. (2011). Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B. Mol Biol Cell 22, 4486-4502. PMID: 21965289

Carmena, M., Pinson, X., Platani, M., Salloum, Z., Xu, Z., Clark, A., Macisaac, F., Ogawa, H., Eggert, U., Glover, D.M., et al. (2012). The chromosomal passenger complex activates Polo kinase at centromeres. PLoS Biol 10, e1001250. PMID: 22291575

Chan, Y. W., Jeyaprakash, A. A., Nigg, E. A., and Santamaria, A. (2012). Aurora B controls kinetochore-microtubule attachments by inhibiting Ska complex-KMN network interaction. J Cell Biol 196, 563-571.

Chen, Z.A., Jawhari, A., Fischer, L., Buchen, C., Tahir, S., Kamenski, T., Rasmussen, M., Lariviere, L., Bukowski-Wills, J.C., Nilges, M., et al. (2010). Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J 29, 717-726. PMID: 20094031

Cheval, H., Guy, J., Merusi, C., De Sousa, D., Selfridge, J., and Bird, A. (2012). Postnatal inactivation reveals enhanced requirement for MeCP2 at distinct age windows. Hum Mol Genet. 2012 Jun 13. [Epub ahead of print].

Choi, E.S., Stralfors, A., Castillo, A.G., Durand-Dubief, M., Ekwall, K., and Allshire, R.C. (2011). Identification of Noncoding Transcripts from within CENP-A Chromatin at Fission Yeast Centromeres. J Biol Chem 286, 23600-23607. PMID: 21531710

Clift, D., and Marston, A.L. (2011). The role of shugoshin in meiotic chromosome segregation. Cytogenet Genome Res 133, 234-242. PMID: 21273764

Clouaire, T., de Las Heras, J.I., Merusi, C., and Stancheva, I. (2010). Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA. Nucleic Acids Res 38, 4620-4634. PMID: 20378711

Cobb, S., Guy, J., and Bird, A. (2010). Reversibility of functional deficits in experimental models of Rett syndrome. Biochem Soc Trans 38, 498-506. PMID: 20298210

Cook, A.G., and Conti, E. (2010). Nuclear export complexes in the frame. Curr Opin Struct Biol 20, 247-252. PMID: 20171875

Cordin, O., Hahn, D., and Beggs, J.D. (2012). Structure, function and regulation of spliceosomal RNA helicases. Curr Opin Cell Biol 24, 431-438. PMID: 22464735

Cortazar, D., Kunz, C., Selfridge, J., Lettieri, T., Saito, Y., MacDougall, E., Wirz, A., Schuermann, D., Jacobs, A.L., Siegrist, F., et al. (2011). Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature 470, 419-423. PMID: 21278727

Davidson, L., Kerr, A., and West, S. (2012). Co-transcriptional degradation of aberrant pre-mRNA by Xrn2. EMBO J 31, 2566-2578. PMID: 22522706

Deaton, A.M., and Bird, A. (2011). CpG islands and the regulation of transcription. Genes Dev 25, 1010-1022. PMID: 21576262

Deaton, A.M., Webb, S., Kerr, A.R., Illingworth, R.S., Guy, J., Andrews, R., and Bird, A. (2011). Cell type-specific DNA methylation at intragenic CpG islands in the immune system. Genome Res 21, 1074-1086. PMID: 21628449

Defossez, P.A., and Stancheva, I. (2011). Biological functions of methyl-CpG-binding proteins. Prog Mol Biol Transl Sci 101, 377-398. PMID: 21507359

Dietrich, N., Lerdrup, M., Landt, E., Agrawal-Singh, S., Bak, M., Tommerup, N., Rappsilber, J., Sodersten, E., and Hansen, K. (2012). REST-mediated recruitment of polycomb repressor complexes in mammalian cells. PLoS Genet 8, e1002494. PMID: 22396653

Dunsmore, C.J., Malone, K.J., Bailey, K.R., Wear, M.A., Florance, H., Shirran, S., Barran, P.E., Page, A.P., Walkinshaw, M.D., and Turner, N.J. (2011). Design and synthesis of conformationally constrained cyclophilin inhibitors showing a cyclosporin-A phenotype in C. elegans. Chembiochem 12, 802-810. PMID: 21337480

El Hage, A., French, S.L., Beyer, A.L., and Tollervey, D. (2010). Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev 24, 1546-1558. PMID: 20634320

Fant, X., Samejima, K., Carvalho, A., Ogawa, H., Xu, Z., Yue, Z., Earnshaw, W.C., and Ruchaud, S. (2010). Use of DT40 conditional-knockout cell lines to study chromosomal passenger protein function. Biochem Soc Trans 38, 1655-1659. PMID: 21118143

Fernandez-Miranda, G., Perez de Castro, I., Carmena, M., Aguirre-Portoles, C., Ruchaud, S., Fant, X., Montoya, G., Earnshaw, W.C., and Malumbres, M. (2010). SUMOylation modulates the function of Aurora-B kinase. J Cell Sci 123, 2823-2833. PMID: 20663916

Flors, C., and Earnshaw, W.C. (2011). Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes. Curr Opin Chem Biol 15, 838-844. PMID: 22098720

Fraser, J.A., Madhumalar, A., Blackburn, E., Bramham, J., Walkinshaw, M.D., Verma, C., and Hupp, T.R. (2010). A novel p53 phosphorylation

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site within the MDM2 ubiquitination signal: II. a model in which phosphorylation at SER269 induces a mutant conformation to p53. J Biol Chem 285, 37773-37786. PMID: 20847049

French, S.L., Sikes, M.L., Hontz, R.D., Osheim, Y.N., Lambert, T.E., El Hage, A., Smith, M.M., Tollervey, D., Smith, J.S., and Beyer, A.L. (2011). Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes. Mol Cell Biol 31, 482-494. PMID: 21098118

Fuad, F.A., Fothergill-Gilmore, L.A., Nowicki, M.W., Eades, L.J., Morgan, H.P., McNae, I.W., Michels, P.A., and Walkinshaw, M.D. (2011). Phosphoglycerate mutase from Trypanosoma brucei is hyperactivated by cobalt in vitro, but not in vivo. Metallomics 3, 1310-1317. PMID: 21993954

Galli, G.G., Honnens de Lichtenberg, K., Carrara, M., Hans, W., Wuelling, M., Mentz, B., Multhaupt, H.A., Fog, C.K., Jensen, K.T., Rappsilber, J., et al. (2012). Prdm5 regulates collagen gene transcription by association with RNA polymerase II in developing bone. PLoS Genet 8, e1002711. PMID: 22589746

Gardano, L., Holland, L., Oulton, R., Le Bihan, T., and Harrington, L. (2012). Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic Acids Res 40, e36. PMID: 22187156

Gendrel, A.V., Apedaile, A., Coker, H., Termanis, A., Zvetkova, I., Godwin, J., Tang, A., Huntley, D., Montana, G., Taylor, S., et al. (2012). Smchd1 dependent and independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome s. Dev Cell In Press. PMID:

Gerlach, B., Kleinschmidt, J.A., and Bottcher, B. (2011). Conformational changes in adeno-associated virus type 1 induced by genome packaging. J Mol Biol 409, 427-438. PMID: 21463638

Grainger, R.J., and Beggs, J.D. (2012). Pre-mRNA splicing. In: Brenner’s Online Encyclopedia of Genetics 2nd Edition, Oxford. In press. PMID:

Granneman, S., Petfalski, E., Swiatkowska, A., and Tollervey, D. (2010). Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking. EMBO J 29, 2026-2036. PMID: 20453830

Granneman, S., Petfalski, E., and Tollervey, D. (2011). A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease. EMBO J 30, 4006-4019. PMID: 21811236

Green, L.C., Kalitsis, P., Chang, T.M., Cipetic, M., Kim, J.H., Marshall, O., Turnbull, L., Whitchurch, C.B., Vagnarelli, P., Samejima, K., et al. (2012). Contrasting roles of condensin I and condensin II in mitotic chromosome formation. J Cell Sci 125, 1591-1604. PMID: 22344259

Guy, J., Cheval, H., Selfridge, J., and Bird, A. (2011). The Role of

MeCP2 in the Brain. Annu Rev Cell Dev Biol 27, 631-652. PMID: 21721946

Hahn, D., and Beggs, J.D. (2010). Brr2p RNA helicase with a split personality: insights into structure and function. Biochem Soc Trans 38, 1105-1109. PMID: 20659012

Hardwick, K.G., and Shah, J.V. (2010). Spindle checkpoint silencing: ensuring rapid and concerted anaphase onset. F1000 Biol Rep 2, 55. PMID: 21173869

Harrington, L. (2012). Haploinsufficiency and telomere length homeostasis. Mutat Res 730, 37-42. PMID: 22100521

Houseley, J., and Tollervey, D. (2010). Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro. PLoS One 5, e12271. PMID: 20805885

Houseley, J., and Tollervey, D. (2011). Repeat expansion in the budding yeast ribosomal DNA can occur independently of the canonical homologous recombination machinery. Nucleic Acids Res 39, 8778-8791. PMID: 21768125

Hsin, K.Y., Morgan, H.P., Shave, S.R., Hinton, A.C., Taylor, P., and Walkinshaw, M.D. (2010). EDULISS: a small-molecule database with data-mining and pharmacophore searching capabilities. Nucleic Acids Res 39, D1042-1048. PMID: 21051336

Husi, H., McAllister, F., Angelopoulos, N., Butler, V.J., Bailey, K.R., Malone, K., Mackay, L., Taylor, P., Page, A.P., Turner, N.J., et al. (2010). Selective chemical intervention in the proteome of Caenorhabditis elegans. J Proteome Res 9, 6060-6070. PMID: 20804218

Iida, Y., Kim, J.H., Kazuki, Y., Hoshiya, H., Takiguchi, M., Hayashi, M., Erliandri, I., Lee, H.S., Samoshkin, A., Masumoto, H., et al. (2010). Human artificial chromosome with a conditional centromere for gene delivery and gene expression. DNA Res 17, 293-301. PMID: 20798231

Illingworth, R.S., Gruenewald-Schneider, U., Webb, S., Kerr, A.R., James, K.D., Turner, D.J., Smith, C., Harrison, D.J., Andrews, R., and Bird, A.P. (2010). Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet 6. PMID: 20885785

Jeyaprakash, A. A., Santamaria, A., Jayachandran, U., Chan, Y, W., Benda, C., Nigg, E. A., and Conti, E. (2012). Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface. Mol Cell 46, 274-286.

Jeyaprakash, A.A., Basquin, C., Jayachandran, U., and Conti, E. (2011). Structural basis for the recognition of phosphorylated histone H3 by the surviving subunit of the chromosomal passenger complex. Structure 19, 1625-1634.

Jorda, R., Havlicek, L., McNae, I.W., Walkinshaw, M.D., Voller, J., Sturc, A., Navratilova, J., Kuzma, M., Mistrik, M., Bartek, J., et al. (2011). Pyrazolo[4,3-d]pyrimidine bioisostere of roscovitine: evaluation of a novel selective inhibitor of cyclin-dependent kinases

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with antiproliferative activity. J Med Chem 54, 2980-2993. PMID: 21417417

Jorda, R., Sacerdoti-Sierra, N., Voller, J., Havlicek, L., Kracalikova, K., Nowicki, M.W., Nasereddin, A., Krystof, V., Strnad, M., Walkinshaw, M.D., et al. (2011). Anti-leishmanial activity of disubstituted purines and related pyrazolo[4,3-d]pyrimidines. Bioorg Med Chem Lett 21, 4233-4237. PMID: 21683592

Kerr, A.R., and Schirmer, E.C. (2011). FG repeats facilitate integral protein trafficking to the inner nuclear membrane. Commun Integr Biol 4, 557-559. PMID: 22046461

Kim, J.H., Kononenko, A., Erliandri, I., Kim, T.A., Nakano, M., Iida, Y., Barrett, J.C., Oshimura, M., Masumoto, H., Earnshaw, W.C., et al. (2011). Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proc Natl Acad Sci U S A 108, 20048-20053. PMID: 22123967

Kirino, Y., Vourekas, A., Kim, N., de Lima Alves, F., Rappsilber, J., Klein, P.S., Jongens, T.A., and Mourelatos, Z. (2010). Arginine methylation of vasa protein is conserved across phyla. J Biol Chem 285, 8148-8154. PMID: 20080973

Kirino, Y., Vourekas, A., Sayed, N., de Lima Alves, F., Thomson, T., Lasko, P., Rappsilber, J., Jongens, T.A., and Mourelatos, Z. (2010). Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization. RNA 16, 70-78. PMID: 19926723

Kleine-Kohlbrecher, D., Christensen, J., Vandamme, J., Abarrategui, I., Bak, M., Tommerup, N., Shi, X., Gozani, O., Rappsilber, J., Salcini, A.E., et al. (2010). A functional link between the histone demethylase PHF8 and the transcription factor ZNF711 in X-linked mental retardation. Mol Cell 38, 165-178. PMID: 20346720

Korfali, N., Srsen, V., Waterfall, M., Batrakou, D.G., Pekovic, V., Hutchison, C.J., and Schirmer, E.C. (2011). A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal. PLoS One 6, e18762. PMID: 21533191

Korfali, N., Wilkie, G.S., Swanson, S.K., Srsen, V., Batrakou, D.G., Fairley, E.A., Malik, P., Zuleger, N., Goncharevich, A., de Las Heras, J., et al. (2010). The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture. Mol Cell Proteomics 9, 2571-2585. PMID: 20693407

Kos, M., and Tollervey, D. (2010). Yeast pre-rRNA processing and modification occur cotranscriptionally. Mol Cell 37, 809-820. PMID: 20347423

Kudla, G., Granneman, S., Hahn, D., Beggs, J.D., and Tollervey, D. (2011). Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast. Proc Natl Acad Sci U S A 108, 10010-10015. PMID: 21610164

Lancaster, O.M., Breuer, M., Cullen, C.F., Ito, T., and Ohkura, H. (2010). The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila. PLoS Genet 6, e1001179. PMID: 21060809

Lando, D., Endesfelder, U., Berger, H., Subramanian, L., Dunne, P.D., McColl, J., Klenerman, D., Carr, A.M., Sauer, M., Allshire, R.C., et al. (2012). Quantitative single molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast. Open Biology 2,120078.

Lebaron, S., Schneider, C., van Nues, R.W., Swiatkowska, A., Walsh, D., Bottcher, B., Granneman, S., Watkins, N.J., and Tollervey, D. (2012). Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nat Struct Mol Biol. PMID: 22751017

Lejeune, E., and Allshire, R.C. (2011). Common ground: small RNA programming and chromatin modifications. Curr Opin Cell Biol 23, 258-265. PMID: 21478005

Lejeune, E., Bayne, E.H., and Allshire, R.C. (2011). On the connection between RNAi and heterochromatin at centromeres. Cold Spring Harb Symp Quant Biol 75, 275-283. PMID:

Libri, V., Helwak, A., Miesen, P., Santhakumar, D., Borger, J.G., Kudla, G., Grey, F., Tollervey, D., and Buck, A.H. (2012). Murine cytomegalovirus encodes a miR-27 inhibitor disguised as a target. Proc Natl Acad Sci U S A 109, 279-284. PMID: 22184245

Liu, Y., and Harrington, L. (2012). Long telomeres bypass the requirement for long telomeres in human tumorigenesis. In: Telomerases: Chemistry, Biology, and Clinical Applications C. Autexier, and N.F. Lue, eds. (New York, John Wiley & Sons). PMID:

Loh, B.J., Cullen, C.F., Vogt, N., and Ohkura, H. (2012). The conserved kinase SRPK regulates karyosome formation and spindle microtubule assembly in Drosophila oocytes. J Cell Sci In press. PMID:

Ludwig, C., Wear, M.A., and Walkinshaw, M.D. (2010). Streamlined, automated protocols for the production of milligram quantities of untagged recombinant human cyclophilin-A (hCypA) and untagged human proliferating cell nuclear antigen (hPCNA) using AKTAxpress. Protein Expr Purif 71, 54-61. PMID: 19995609

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Wellcome Trust Four Year PhD Programmein Cell Biology

Wellcome Trust Centre for Cell BiologyUniversity of Edinburgh

The four year PhD programme at the Centre for Cell Biology offers training in cell biology from outstanding researchers in a stimulating environment. Five places are available each year by competitive interview.

The first year of the programme combines mini-projects with taught courses which cover a wide range of techniques important for modern cell biology including advanced microscopy, molecular biology, proteomics, bioinformatics and systems biology. At the end of the first year, the degree of MSc by Research is awarded to qualifying students, based on rotation project reports and a PhD project outline. This is followed by three further years of full time research.

Funding from the Wellcome Trust for this programme includes a generous student stipend and payment of tuition fees (at the EU rate).

For further information and application form visit www.wcb.ed.ac.uk/phd

Application forms will be available in October.

Application deadline: Wednesday, 12th December 2012

International Scientific Advisory Board

Angelika AmonCenter for Cancer ResearchHoward Hughes Medical InstituteMassachusetts Institute of Technology40 Ames StreetCambridge MA 02139

Frank GrosveldDepartment of Cell BiologyErasmus Medical CenterDr Molewaterplein 503000 RotterdamNetherlands

Michael RoutThe Rockefeller University1230 York AvenueNew York, NY 10021USA

Eric KarsentiEuropean Molecular Biology LaboratoriesMeyerhofstraße 169117 HeidelbergGermany

Nick ProudfootSir William Dunn School of PathologyUniversity of OxfordSouth Parks RoadOxford OX3

Wellcome Trust Centre for Cell BiologySchool of Biological SciencesThe University of Edinburgh

Michael Swann BuildingMayfield Road

Edinburgh EH9 3JRScotland, UK

Telephone +44 (0)131 650 7005Fax +44 (0)131 650 4968

Website www.wcb.ed.ac.uk

Brochure designed by Graphics Lab, Learning Technology Section, The University of Edinburgh.Cover image: “Trapped transcription” (wire and glass) by Sarah Keer-Keer.

The work was inspired by EM images of “Miller” chromatin spreads that reveal nascent pre-rRNA transcripts emanating from the central rDNA strand.

The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336