Nuclear RNA Surveillance & Turnover David Bedwell Post-Transcriptional Regulatory Mechanisms MIC759...

Nuclear RNA Surveillance & Turnover

David Bedwell

Post-Transcriptional Regulatory Mechanisms

MIC759

Sept. 12, 2006

mRNA quality control

• Multiple processing and localization steps are required for proper mRNA maturation following the synthesis of the primary transcript.

• At a minimum, these include capping, splicing, 3´-end cleavage, polyadenylation, and nuclear export.

• If each of these 5 processes are 90% efficient, only ~59% of mRNAs will be properly matured.

• What happens to the improperly processed transcripts?

– Do they get sent to the cytoplasm for protein synthesis anyway?

– Do they get degraded before they can make potentially defective proteins?

Section I. Nuclear mRNA Turnover pathways

mRNA synthesis involves a series of processing steps to synthesize a mature transcript that is proofread in parallel by nuclear surveillance mechanisms at each step.

The splicing and processing components as well as export factors are co-transcriptionally recruited to the nascent transcript, possibly by the transcription complex, and the correct mRNA forms the core of an export-competent mRNP that is eventually exported and expressed in the cytoplasm.

Alternatively, aberrant synthesis or processing leads to retention of the defective transcript. These retained transcripts subsequently undergo exosome-mediated degradation.

Nuclear mRNA surveillance occurs at checkpoints during maturation

Enzyme Activity

Rrp6p 3´5´ exoribonuclease (nuc)

Core exosome 3´5´ exoribonucleases (nuc & cyto)

Mtr4p Putative RNA helicase (nuc)

Rat1p 5´3´ exoribonuclease (nuc)

Xrn1p 5´3´ exoribonuclease (cyto)

Dcp1p Decapping enzyme (nuc & cyto)

Dcp2p Decapping enzyme (nuc & cyto)

Lsm2-8p Stimulates nuclear decapping

Ccr4p/Pop2p poly(A)-specific exoribonuclease (cyto)

Pan2p/Pan3p poly(A)-specific exoribonuclease (nuc & cyto)

Saguez et al., Curr Opin Cell Biol 17: 287-293 (2005)

Nuclear degradation activities in yeast

3´5´ turnover

5´3´ turnover

decapping

deadenylases

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Butler, Trends in Cell Biol 12: 90-96 (2002)

Substrates, components and products of the nuclear exosome

10 core exosome subunits

Putative exonucleasesDemonstrated exonucleases

Mtr4p helicase

degradation

3´-end formation




Nuclear and Nucleolar distribution of Rrp6p using Rrp6p-GFP fusion protein in yeast cells

a) Visible field (DIC)

b) DNA strain for nucleus (Hoescht)

c) GFP-Rrp6p

d) Overlay of GFP-Rrp6p and DNA



Anderson, Curr Biol 15: R635-R638 (2005)

The TRAMP complex polyadenylates RNAs to trigger their degradation

TRAMP complex:

- Trf4p: poly(A) polymerase- Air2p: RNA binding protein- Mtr4p: ATP-dependant RNA helicase

- Trf5p is highly related to Trf4p- Air1p is highly related to Air2p

- The TRAMP complex interacts with RNAs or RNP complexes, making them targets for degradation. For most substrates other than tRNAs, this will primarily be via protein:protein interactions.

-The zinc finger domains of Air2p might be involved in substrate binding. The RNA is then polyadenylated by Trf4p.

- Exosome recruitment and activation requires the intact TRAMP complex. The activated exosome then rapidly deadenylates the RNA and can penetrate into regions of stable structure.

- Helicase activity of Mtr4p is important for dissociation or remodeling of stable RNP structures to allow passage of the exosome.



Roles of the TRAMP Complex in RNA Degradation

LaCava et al., Cell 121: 713-724 (2005)



Anderson, Curr Biol 15: R635-R638 (2005)

Proposed model for degradation of poly(A)+ tRNA by the exosome

- defective tRNA polyadenylated by Trf4p of the TRAMP complex, which stimulates degradation by exosome.

- exosome may stall at highly structured regions, leaving an intact but shortened tRNA (tR).

- tR undergoes another round of polyadenylation and degradation by the exosome.

- cycle continues until tRNA completely degraded.

Model for quality control of stable RNA synthesis in bacteria. Genes encoding normal (wt-tRNA) and defective (ts-tRNA) RNAs are transcribed with equal efficiency to generate tRNA precursors. However, whereas wt-tRNA precursor is rapidly converted to its mature form by the processing RNases, maturation of the defective precursor is greatly slowed. As a consequence, the defective tRNA precursor is first subject to polyadenylation by poly(A) polymerase, and is then degraded by PNPase and other RNases.



Polyadenylation is also involved in stable RNA degradation in bacteria

Li et al., EMBO J 21: 1132-1138 (2002)




Substrates, components and products of the cytoplasmic exosome

Section II. Nuclear surveillance occurs at various nuclear

checkpoints and is coupled to transcription, mRNA processing

and mRNA export



Aguilera, Curr Opin Cell Biol 17: 242-250 (2005)

Eukaryotic gene expression includes:

- transcription - nuclear mRNP processing steps

- 5´-end capping- splicing- mRNP assembly- 3´-end cleavage- polyadenylation

- mRNP surveillance- RNA export

All of these steps are coupled or coordinated with transcription.

NMD, nonsense-mediated decay; NPC, nuclear pore complex; RNAPII, RNA polymerase II.

Simplified view of eukaryotic gene expression



Vasudevan and Peltz, Curr Opin Cell Biol 15: 332-337 (2003)

Overview of nuclear mRNA surveillance

Exosome

Exosome

RNA Polymerase II

• RNA polymerase II (also called RNAP II or Pol II) transcribes DNA to synthesize precursors of mRNAs and most snRNAs.

• A 550 kDa complex of 12 subunits. A wide range of transcription factors are required for it to bind to its promoters and begin transcription.

• The largest subunit of Pol II (Rpb1) has a domain at its C-terminus that is called the CTD. Phosphorylation of the CTD is an important regulation mechanism, as this allows the binding and release of many factors that influence not only the transcription process, but also mRNA maturation and export from the nucleus. In this way, the CTD provides a platform for various factors that load on the nascent mRNA chain during transcription.

• The CTD consists of heptapeptide repeats (consensus: YSPTSPS) ranging from 26 in yeast and 52 in mammals, out of which serines and threonines get phosphorylated.

• Patterns of phosphorylation on these repeats can change rapidly during transcription. The regulation of the phosphorylation pattern and the resulting differential association of factors plays a major role not only in the regulation of transcription, but also in the fate of mRNA transcripts.



Bentley, Curr Opin Cell Biol 17: 251-256 (2005)

Capping enzymes (guanylyltransferase [GT] and 7-methyltransferase [MT]), cap binding complex (CBC), cleavage/ polyadenylation factors (C/P), and the RNA 5´3´ exonuclease, Rat1, are indicated.

Processing factors interact with pol II elongation complex via the CTD (green line) and probably also via the nascent RNA (red line). Phosphorylation of Ser2 and Ser5 residues in the CTD heptad repeats are marked P2 and P5. Capping enzymes stimulate early steps in pol II transcription including promoter clearance denoted by the thick arrow. Following addition of the cap, MeGppp, GT is released from the elongation complex.

Co-transcriptional recruitment of pre-mRNA processing factors at the 5´ end of a gene



Bentley, Curr Opin Cell Biol 17: 251-256 (2005)

Co-transcriptional splicing and cleavage/polyadenylation

The spliceosome is recruited to intron RNA (black line) and probably also to the pol II elongation complex, at least in metazoans. Co-transcriptional splicing releases the intron (black lariat). Note that while spliceosome assembly probably occurs co-transcriptionally on most introns, excision of the intron can occur post-transcriptionally. More cleavage/polyadenylation factors (C/P) and Rat1 are detectable at the 3´ end than at the 5´ end of the gene. These factors interact with the nascent RNA at the poly(A) site, AAUAAA, to cleave and polyadenylate the transcript. The Rat1 exonuclease degrades RNA downstream of the cleavage site (scissors) and helps trigger termination of transcription.



Luo et al., Genes & Development 20: 954-965 (2006)

The “allosteric torpedo” model couples transcription termination with 3´-end cleavage

- Association of Rat1 and cleavage/polyadenylation factors exemplified by Pcf11 with the CTD Ser2 phosphorylated pol II elongation complex is shown at the poly(A) site (AATAAA).

-This interaction results in an allosteric change in pol II (designated by a change from green to red) that favors termination. (Blue Ps) Ser5-PO4; (red Ps) Ser2-PO4.

- Nascent RNA downstream of the poly(A) cleavage site (blue line) is degraded by both Xrn1 and Rat1. This degradation could facilitate termination; however, it is not sufficient to cause termination.

THO Complex

• THO was identified as a four protein complex containing proteins encoded by THO2 and HPR1, two genes previously identified by hyper-recombination mutations, and MFT1 and THP2. The null mutations of each of the four genes are viable and yield similar phenotypes of impairment of transcription elongation and transcription-dependent hyper-recombination between direct repeats.

• It was previously proposed that the THO complex has a functional role related to RNAP II transcription elongation. Whether this role is direct or indirect is not yet known. However, some studies suggest that THO might also have a role in RNA metabolism beyond transcription.

• Recent studies have shown that THO and RNA export factors are functionally related. For example, multicopy Sub2p suppresses the transcriptional defect of hpr1 cells, and sub2, yra1, mex67 and mtr2 mutants show similar defective transcription and hyper-recombination phenotypes to THO mutants.

The TREX complex contains THO and 2 mRNA export factors

• In yeast, the multi-subunit TREX complex plays a role in coupling

transcription to mRNA export. This complex contains the mRNA export factors Sub2p and Yra1p as well as the THO complex, which functions in transcription elongation.

• Human TREX contains Aly/REF, UAP56, and the human

counterpart of the yeast THO complex. The human THO complex specifically associates with spliced mRNA and not with unspliced pre-mRNA.

• Recent data indicate that recruitment of the human TREX complex to spliced mRNA occurs by a splicing-coupled mechanism rather than by the direct transcription-coupled

mechanism that occurs in yeast.

• Deletion mutants of THO components show defects in

transcription elongation and have a hyper-recombination phenotype. The elongation defects and hyper-recombination appear to be due to the presence of RNA/DNA hybrids that form between the nascent RNA and the DNA template.



The THO complex in yeast is recruited co-transcriptionally to nascent mRNAs through interactions with factors implicated in transcription elongation. This leads to the recruitment of Sub2p and Yra1p (UAP56 and REF1/Aly in higher eukaryotes) forming the TREX complex.

Properly assembled and processed mRNPs are released in the nucleoplasm and proceed to subsequent steps of mRNP export. Either delay or impairment of mRNP assembly and/or 3´-end processing would result in the retention of the transcripts near or at the site of transcription (pale orange area), providing a window of opportunity for the exosome to act.

It is currently unknown whether binding of Mex67p–Mtr2p (NXF1–p15 in higher eukaryotes) occurs in a co- or post-transcriptional manner. The association of Mex67p-Mtr2p may displace Sub2p just before NPC-translocation.

Co-transcriptional recruitment of mRNA export

factors in yeast

Stutz & Izaurralde, Trends in Cell Biol 13: 319-327 (2003)

Do the THO & TREX complexes really function in transcription elongation?

• Deletion mutants of THO components show defects in

transcription elongation (particularly 3´-truncated transcripts) and have a hyper-recombination phenotype. The elongation defects

and hyper-recombination appear to be due to the presence of

RNA/DNA hybrids that form between the nascent RNA and the DNA template.

• However, hyper-recombination and transcription defects are also observed with mutations in Sub2 and Yra1, as well as with mutations in other proteins involved in mRNA export, including Sac3, Thp1, Mex67, Mtr2, and Nab2.

• Furthermore, the human THO complex specifically associates with spliced mRNA and not with unspliced pre-mRNA. This suggests that recruitment of the human TREX complex to spliced mRNA occurs by a splicing-coupled mechanism rather than by the direct transcription-coupled mechanism that occurs in yeast.

Vinciguerra & Stutz, Curr Opin Cell Biol 16: 285-292 (2004)



Model 1: The exosomal model proposes that co-transcriptional surveillance by the nuclear exosome results in the retention and 3´5´ degradation of malformed mRNPs at or close to the site of transcription. In this view, transcripts are fully synthesized but unstable and degraded.

Model 2: The transcriptional model claims that malformed mRNP complexes form stretches of DNA:RNA hybrids with the coding DNA strand. The DNA:RNA hybrids impair transcription elongation by creating an obstacle for the next elongating RNA polymerase.

Both views propose that an important role of THO/TREX is to promote efficient mRNP assembly, preventing the formation of DNA:RNA hybrids during elongation and protecting the mRNP from degradation by the exosome.

Non-exclusive models to explain low levels of 3´-truncated transcripts in TREX mutants

Section III. nuclear mRNA export receptors

The hodgepodge of factors that influence mRNA export


Yeast Metazoans

Mex67p NXF1 (TAP)

Mtr2p NXT1 (p15)

Npl3p ?

Nab2p ?

Sub2p UAP56 (Hel)

Yralp Aly (Ref1)

Yra2p Ref2

Tex1p Tex1

Tho2p Tho2p

Hpr1p Hpr1p

Mft1p ?

Thp2p ?

THOComplex

TREXComplex

mRNA export

receptor

mRNA export

adaptors




Phylogenetic tree of NXF (TAP) family sequences

- Tree was drawn by the neighbor-joining method, using the most conserved part of NXF protein sequences (this corresponds to residues 212 to 539 of the Hs NXF1 sequence).

- The phylogenetic tree indicates that separate gene-duplication events have occurred in several eukaryotic lineages.

- Although higher eukaryotes have several NXF homologs, they evolved independently so that there is no clear one-to-one relationship between isoforms.

Abbreviations: Ag, Anopheles gambiae; Cc, Coturnix coturnix (quail); Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.



Factors involved in Nuclear Export of mRNA

Cullen, J. Cell Sci 116: 587 (2003)

(Sub2p)

(Yra1p) (Mtr2p)

(Mex67p)

(Corresponding yeast factors indicated in parentheses)



Linder & Stutz, Curr Biol 11: R961-963 (2001)

Sub2p/UAP56, Yra1p/Aly/REF and hnRNP proteins such as Npl3p associate co-transcriptionally with the mRNA (CBC is the cap-binding complex). In the case of intron-containing genes, the spliceosome also assembles on the pre-mRNA.

In mammalian cells, Aly/REF and UAP56 are part of the exon-junction complex on the spliced mRNA (not shown). The Sub2p/UAP56 protein is replaced by the Mex67p–Mtr2p/TAP-p15 heterodimers, which mediate the interaction of the mRNP with components of the nuclear pore complex (NPC).

The DEAD box protein Dbp5p is required for release of mRNP on the cytoplasmic side of the NPC. DEAD box-mediated ATPase activities important for mRNA export are indicated by stars.

The mRNA ‘relay race’ from the site of transcription to the delivery to ribosomes in the cytosol




Mex67p and NXF1 are essential for export of bulk mRNA

Shifting the mex67ts strain to the restrictive temperature (37°C) causes

nuclear mRNA accumulation.



Depletion of Drosophila cells of NXF1 by double stranded RNA inhibition causes

nuclear mRNA accumulation.

In both experiments, poly(A) mRNA was visualized by in situ hybridization with a

fluorescently-labeled oligo-dT probe



At steady state, non-heat shock (non-hs) genes are responsible for the majority of transcription. These mRNAs are bound by RNA binding proteins, such as the cap binding complex (CBC), the hnRNP protein Npl3, and many other proteins not indicated (black circles).

Yra1 promotes the association of Mex67 with the RNP, and this complex is directed to and exported through the NPC via interactions with Mtr2 and nucleoporins. The helicase Dbp5 may strip RNA binding proteins off the mRNA to allow these proteins to return to the nucleus and translation factors to associate with the mRNA and initiate translation.

Lei & Silver, Dev. Cell 2: 261-272 (2002)

Regulated mRNA Export during Heat Shock



In response to heat shock, non-hs genes are repressed, and transcription of heat shock genes is greatly stimulated. Npl3 disassociates from bulk mRNA and may cause non-hs RNPs to become export incompetent. Heat shock mRNAs may preferentially recruit mRNA export factors, such as Mex67, to bypass retention mechanisms.

Lei & Silver, Dev. Cell 2: 261-272 (2002)

Regulated mRNA Export during Heat Shock (cont)

Non-mRNA Export from the Nucleus

Cullen, J. Cell Sci 116: 587 (2003)

VA RNA represents

MicroRNAs

PHAX = Phosphorylated

Adaptor for RNA Export



- The Ran-GTP gradient governs the directionality of nucleocytoplasmic transport mediated by members of the karyopherin family of nuclear transport factors.

-The key role played by the GTP-bound form of Ran in mediating cargo binding and release by karyopherins functioning in nuclear import (importins) or nuclear export (exportins) is illustrated.

- Imp, importin; Exp, exportin.

- NOTE: the Ran system is not involved in mRNA export using the NXF (TAP) export receptors.

The Ran-GTP gradient governs the directionality of some forms of nucleocytoplasmic transport

Cullen, J. Cell Sci 116: 587 (2003)Ran-GAP located in

cytoplasmRan-GEF located in

nucleus

Section IV. A nuclear surveillance checkpoint is also present at the

nuclear pore

Mlp proteins (Mlp1 and Mlp2) may play a general role in mRNP surveillance by preferentially interacting with properly packaged mRNP complexes, preventing mRNPs that lack essential signals from reaching the central channel of the NPC. The Mlp proteins are not essential for viability.

A quality control checkpoint at the nuclear pore is mediated by Mlp proteins




The perinuclear Mlp1p protein contributes to mRNP surveillance by retaining unspliced transcripts within the nucleus, possibly via recognition of a component associated with the 5´ splice site.

Mlp proteins form a selective filter at the entrance of the nuclear pore complex




Nab2p, a shuttling mRNA binding protein involved in polyA tail length regulation, directly interacts with Mlp proteins.

Nab2p may be important for the docking of mRNPs to the Mlp barrier, perhaps by signaling proper 3´ end formation.

Nab2p mediates the Mlp checkpoint at the entrance of the nuclear pore complex



- In wild type cells, the mRNA-binding proteins Yra1p and Nab2p are essential for mRNP docking to the Mlp export gate at the nuclear periphery.

- mRNP complexes produced in the GFP-yra1-8 mutant strain are retained by the Mlp selective filter and mRNP stalling negatively feeds back on mRNA synthesis.

- Loss of Mlp1p or Mlp2p alleviates the negative effect on mRNA synthesis and allows a fraction of transcripts to reach the cytoplasm.

Mlp proteins establish a link between mRNA synthesis and export

Vinciguerra et al., EMBO J 24: 813-823 (2005)

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Nuclear RNA Surveillance & Turnover David Bedwell Post-Transcriptional Regulatory Mechanisms MIC759...

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