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from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 (1999)
Homology-dependent Gene Silencing – The World in 1999
TGS – Pairing of tightly linked homologous loci induces methylationTranscriptional Gene Silencing
PTGS – Transcript-specific degradationPost-transcriptional Gene Silencing
SAS – Spread of PTGSSystemic Acquired Silencing
RIP – Induction of C-T transitionsRepeat-induced Point Mutation
RNAiRNA interference
from tenOever, Nature Rev.Microbiol. 11, 169 (2013)
Response to Virus Infection in Chordates
Viral dsRNA is recognized by PRRs in the cytoplasm or TLRs in endosomes
Induce expression of type I interferons
Leads to transactivation of >250 genes
Slows viral infection and allows time for an adaptive immune response
from tenOever, Nature Rev.Microbiol. 11, 169 (2013)
viRNAs are an Antiviral Innate Immune System
viRNAs are derived from the virus and loaded onto the RISC
viRNAs bind the viral RNA target with perfect complementarity and eliminates the target
Chordates do not produce viRNA
from McManus and Sharp, Nature Rev.Genet. 3, 737 (2002)
Response of Mammalian Cells to Long dsRNA
Long dsRNA induces interferon response in vertebrates
PKR phosphorylates eIF2 to inhibit translation
2’-5-oligoadenylate synthase is induced, which activates RNaseL and leads to nonspecific mRNA degradation
siRNA does not invoke the interferon response
The lin-14 Mutant has an Altered Pattern of Cell Division
The PNDB neuroblast is generated prematurely
The LIN-14 protein prevents L2-type cell divisions
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 21-6
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 21-6
The LIN-14 protein prevents L2-type cell divisions
During L2, lin-4 miRNA prevents translation of lin-14 mRNA
In the adult, let-7 inhibits lin-14 and lin-41 translation
Absence of LIN-41 permits lin-29 translation and generation of adult cell lineages
miRNAs Regulate Development in C. elegans
from Li and Hannon, Nature Rev.Genet. 5, 522 (2004)
lin-4 Inhibits Translation of lin-14 mRNA
Mutations in lin-4 disrupt regulation of larval development in C. elegans
lin-4 antagonizes lin-14 function
lin-4 encodes the precursor to a 22 nt-long microRNA that is partially complementary to sites in the 3’UTR of lin-14 mRNA
Annealing of lin-4 to lin-14 mRNA inhibits translation
from Li and Hannon, Nature Rev.Genet. 5, 522 (2004)
Biogenesis of miRNAs and siRNAs
miRNAs are genomically encoded
siRNAs are produced exogenously or from bidirectionally transcribed RNAs
miRNAs have imperfect complementarity to their target mRNA and inhibit translation
siRNAs form perfect duplex with their target mRNA and trigger mRNA degradation
Drosha processes pri-miRNA to pre-miRNA in the nucleus
miRNA is selectively incorporated into the RISC for target recognition
Guide strand of siRNA is incorporated into the RISC for target recognition
Strand Selection Into the RISC
The strand with its 5’-terminus at the less stable end of the duplex is incorporated into the RISC
from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127 (2005)
Strand Selection of Processed siRNA into the RISC
from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127 (2005)
The PAZ domain of Dicer binds to the pre-existing dsRNA end
The strand that has its 3’-end bound to the PAZ domain preferentially assembles into the RISC
Guide RNA Loading Onto Argonaute
PAZ domain binds 3’-overhang
5’-end of guide RNA is anchored in a conserved pocket of the PIWI domain
Argonaute slices passenger strand of siRNA
from Parker and Barford, Trends Biochem.Sci. 31, 622 (2006)
Mechanisms of miRNA Sequence Diversification
Seed shifting that results from variations in Drosha or Dicer processing generates isomiRs
In arm shifting, mutations within the precursor change the ratio of miRNA to miRNA* loading
In hairpin shifting, the folding is changed into a new configuration
In cells containing adenosine deaminase, A is converted to I
from Berezikov, Nature Rev.Genet. 12, 846 (2011)
The Fate of mRNA Loaded With the miRISC
Targeted mRNA accumulates in P bodies
mRNA is stored in P bodies, undergoes degradation, or reenters the translation pathway
from Rana, Nature Rev.Mol.Cell Biol. 8, 23 (2007)
Role of Poly(A) and Cap in Translation Initiation
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
The cap structure is recognized by eIF4F
Poly(A) is recognized by PABPC
PABPC interacts with eIF4G
Recruitment of the preinitiation complex is increased
miRNAs Promote mRNA Deadenylation
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
miRNA guide strand associates with AGO
AGO interacts with GW182
GW182 may compete with eIF4G for binding to PABPC and prevents mRNA circularization
Assembly of AGO-GW182-PABPC complex triggers deadenylation by CAF1-CCR4-NOT
GW182 may reduce the affinity of PABPC for the poly(A) tail
Fate of Deadenylated mRNAs
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
Deadenylated mRNAs are stored in a translationally repressed state
Deadenylated mRNAs are decapped by DCP2 associated with decapping activators
Decapped mRNA is degraded by XRN1
Overview of RNA-Mediated Gene Silencing
from Eulalio et al., Nature Rev.Mol.Cell Biol. 8, 9 (2007)
siRNA triggers endonucleolytic cleavage of perfectly-matched complementary targets
The resulting mRNA fragments are degraded
miRNA triggers accelerated deadenylation and decapping of partially-complementary targets and requires Argonaute proteins and a P-body component
Cleavage is catalyzed by Argonaute proteins
miRNA represses translation
siRNA
miRNA
Secretion of miRNAs
from Chen et al., Trends Cell Biol. 22, 125 (2012)
Specific miRNAs can be preferentially sorted into vesicles and delivered to recipient cells
Regulation of siRNA Levels in C. elegans
from Timmons, BioEssays 26, 715 (2004)
RNA-dependent RNA polymerase amplifies siRNA
RRF-3 prevents siRNA amplification
ERI-1 is an siRNA-specific RNase
At least 1400 miRNA-encoding genes in humans
miRNAs regulate ~50% of the human transcriptome
Prevalence of and Regulation by miRNAs
miRNAs fine tune the expression of proteins in a cell
from Technau, Nature 455, 1184 (2008)
miRNA complexity correlates with an increase in morphological complexity
Number of protein-coding genes are similar in animals
Organismal Complexity May Be Due to Differences in Regulation of Gene Expression
There is a continuous acquisition of novel miRNAs during evolution
Lineage-specific loss of miRNAs also occurs
There are now estimated to be 1,424 miRNAs in humans
Mutations in heterochronic genes cause temporal cell fate transformations that are altered relative to the timing of events in other cells or tissues
let-7 mutations cause an overproliferation of seam cells
Overproliferation of cells is a characteristic of stem cells and cancer
let-7 is a Heterochronic Gene in C. elegans
from Büssing et al., Trends Mol.Med. 14, 400 (2008)
from Viswanathan and Daley, Cell 140, 445 (2010)
Regulation of Differentiation by let-7
let-7 levels are reduced in stem cells
Lin28 promotes reprogramming by inhibition of let-7 maturation
Reprogramming to iPS Cells
Oct4Sox2Klf4c-Myc
Oct4Sox2NANOGLin28
or
Lin28 represses let-7
Is let-7 repression important for establishment of pleuripotent state?
c-Myc is a let-7 target, so Lin28 replaces c-Myc
Transfection of ESCC (ES cell-specific cell cycle-regulating) miRNAs can generate ES cells without protein-encoding factors
Links of let-7/Lin28 to Cancer
let-7 is a tumor suppressor
The oncogenes c-Myc, K-Ras, and cyclin D1 are let-7 targets
Lin28 is an oncogene that is activated in 15% of human tumors
Lin28 is also a let-7 target
let-7 Lin28
double-negative feedback loop
Lin28 Prevents let-7 Maturation
from Thornton and Gregory, Trends Cell Biol. 22, 474 (2012)
Lin28a and Lin28b repress let-7 biogenesis by two distinct mechanisms
Lin28a recruits TUTase which uridylates the miRNA and promotes let-7 degradation
Lin28b inhibits Drosha-mediated processing of let-7
let-7 promotes differentiation
During differentiation, let-7 targets Lin28 mRNA, which reinforces developmental commitment
from Thornton and Gregory, Trends Cell Biol. 22, 474 (2012)
Lin28 prevents let-7 muturation
let-7 promotes differentiation and prevents transformation
Lin28 promotes reprogramming or transformation
ESCC miRNAs maintain Lin28 expression
Summary of Lin28 let-7 Regulation of Differentiation and Oncogenesis
A MicroRNA Regulates Neuronal Differentiation by Controlling Alternative Splicing
miR-124 targets a component of a repressor of neuron-specific genes
miR-124 results in reduced expression of PTBP1 leading to the accumulation of PTBP2
PTBP2 results in a global switch to neuron-specific alternative splicing patterns
from Makeyev et al., Mol.Cell 27, 435 (2007)
The Role of miRNA in Cancer
miRNA profiles define the cancer type better than mRNA expression data
miRNA expression is lower in cancers than in most normal tissues, but expression of some miRNAs is increased
c13orf25 miRNA is the first non-coding oncogene, is upregulated by c-Myc, and is involved in leukemia development
c13orf25 inhibits expression of E2F1, a cell cycle regulator
The undifferentiated state of malignant cells is correlated with a decrease in miRNA expression
from He et al., Nature 435, 828 (2005) Lu et al., Nature 435, 834 (2005) Lujambio and Lowe, Nature 482, 347 (2012)
Down-regulation of all miRNAs enhanced tumor growth
Loss of miR-126 and miR-355 when human breast cancer cells develop metastatic potential
Restoring expression of these miRNAs in malignant cells suppresses metastasis in vivo
miR-355 targets the progenitor cell transcription factor SOX4, and the ECM component tenascin C
miRNAs and Breast Cancer Metastasis
from Tavasoie et al., Nature 451, 147 (2008)
miR-10b and miR-9 induce metastasis
EZH2 (a PcG protein) overexpression promotes cell proliferation
Expression of EZH2 is inhibited by miR-101
miR-101 expression decreases during prostate cancer progression
Role of MicroRNAs and Epigenetics in Cancer
from Varambally et al., Science 322, 1695 (2008)
miR-29 inhibits DNMT3A and DNMT3B in lung cancer
from Lujambio and Lowe, Nature 482, 347 (2012)
Inhibition of Endogenous miRNA function
Vectors express multiple copies of miRNA target sites
miRNA sponges
Endogenous miRNA is saturated and prevented from silencing its natural product
from Brown and Naldini, Nature Rev.Genet. 10, 578 (2009)
Pseudogene transcripts can act as miRNA sponges
Competitive Endogenous RNAs (ceRNAs)
The human transcriptome contains 21.000 protein-coding genes, 9,000 small RNAs, 10,000-32,000 lncRNAs and 11,000 pseudogenes
70-90% of the human genome is transcribed, but less than 2% of the genome encodes protein-coding genes
All RNA transcripts that contain miRNA binding sites that regulate each other by competing for shared miRNAs
ceRNAs can fine-tune gene expression
The expression level of PTEN is crucial for its tumor suppressive function
PTEN expression is downregulated by miRNAs
PTENP1 is a pseudogene which contains the same MRE in the 3’-UTR
Regulation of PTEN Levels by a Pseuodogene
PTENP1 RNA is a ceRNA that enhances PTEN expression by competing for a shared miRNA
from Rigoutsos, Nature 465, 1016 (2010)
The PTEN ceRNA Network
from Tay et al., Nature 505, 344 (2014)
PTEN expression levels are regulated by a large network of miRNAs, mRNAs, and ceRNAs
The PTEN ceRNA interactions are part of a regulatory layer comprising of more than 248,000 miRNA-mediated interactions
Circular RNAs can be microRNA Sponges
Human fibroblasts have 25,000 circRNAs derived from 15% of transcribed genes
The splicing machinery is involved in circRNA biogenesis
circRNAs are resistant to degradation triggered by miRNAs
from Wilusz and Sharp, Science 340, 440 (2013)
Immunostimulatory Effects of dsRNA
from Kim and Rossi, Nature Rev.Genet. 8, 173 (2007)
Long dsRNA induces PKR
Toll-like receptors in endosomes recognize dsRNA and activate the interferon response
Blunt-ended dsRNA are recognized by RIG-1 helicase and activates the immune response
from Mittal, Nature Rev.Genet. 5, 355 (2004)
The Design of Optimal siRNAs
21 nt RNA that contains 2 nt 3’-overhangs and phosphorylated 5’-ends
Lower stability at the 5’-end of the antisense terminus
Low stability in the RISC cleavage site
Low secondary structure in the targeted region of the mRNA
from Dykxhoorn and Lieberman, Cell 126, 231 (2006)
Delivery of siRNA for Therapy
siRNA is not taken up by most mammalian cells
Cholesterol-conjugated siRNA is taken up by the LDL receptor
siRNA bound to targeted antibody linked to protamine can achieve cell-specific siRNA delivery
from Rossi et al., Nature Biotechnol. 23, 682 (2005)
Fuse Fab targeting antibody with protamine
siRNA binds noncovalently with protamine
Complex is endocytosed into cells expressing the epitope
siRNA is released from the endosome and enters the RISC
Cell-Specific Delivery of siRNA
RNAi-dependent Chromatin Silencing in S. pombe
Overlapping RNAs from centromeric region is processed into siRNA
siRNA activates or recruits Clr3 methyltransferase that methylates H3 on K9
Deletion of RNAi pathway genes cause loss of silencing at centromeres and reduced H3 K9 methylation at centromeric regions
from Allshire, Science 297, 1818 (2002)
Small RNAs Modulate Viral Infection
Viral-encoded miRNA facilitate viral infection and persistence
Viral suppressors of RNA silencing (VSR) inhibit the RNAi pathway
Host cell-encoded miRNAs inhibit or facilitate viral replication
from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006)
SV40 miRNA is synthesized late in the viral life cycle and targets TAg mRNA
SV40 miRNA aids immune invasion by reducing susceptibility to lysis by CTLs
Function of SV40 miRNA
Polyomaviruses also have viral miRNA that targets TAg
Infection with Py mutant lacking the miRNA resulted in no difference in viral load or immune response
from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006)
Effects of Adenovirus VA1 MicroRNA
VA1 binds to and prevents PKR activation to inhibit the innate immune response
VA1 competes with exportin-5 and inhibits Dicer to inhibit the RNAi pathway
from Gupta et al., Nature 442, 82 (2006)
LAT is the only viral gene expressed during latent infection in neurons
miR-LAT is generated from the LAT gene
A MicroRNA was Thought to Protect HSV-1-infected Neurons from Apoptosis
miR-LAT downregulates TGF- and SMAD3 and contributes to the persistence of HSV-1 in neurons in a latent form
Paper retracted – 2008. Repeatedly unable to detect miRNA
from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006)
Cellular miRNAs Modulates Viral Infection
Tas is a PFV-1-encoded protein that inhibits RNAi
miR-32 inhibits viral replication
PFV-1 replication is stimulated by a plant VSR implicating the role of small RNAs in the viral life cycle
miR-122 increases HCV replication in the liver
miR-122 stabilizes the HCV genome by binding the 5’-UTR
miR-122 Protects the HCV Genome From Degradation
from Garcia-Sastre and Evans, Proc.Nat.Acad.Sci. 110, 1571 (2013)
Xrn1 is a cytoplasmic exonuclease that normally degrades HCV RNA
miR-122 increases HCV RNA stability by shielding the genome against Xrn1
miR-122 also enhances HCV RNA replication that is independent on its action against Xrn1
Most miRNAs are transcribed by pol II and processed by Drosha in the nucleus
MHV68 pri-miRNA is transcribed by pol III and processed by tRNase Z
BLV miRNA is transcribed by pol III
miRNA Encoded by an RNA Virus
from Cullen, Proc.Nat.Acad.Sci. 109, 2695 (2012)
PIWIs and piRNAs are enriched in the germline
PIWI mutations result in infertility
piRNA-PIWI pathway is involved in transposon silencing
PIWI depletion results in an upregulation of transposon mRNA expression
PIWIs are expressed in some somatic cells and is important for stem cell function and regeneration in planarians
The Drosophila PIWI phenotype – P-element-induced wimpy testis
Features of piRNAs
Piwi and Aubergine complexes contain piRNAs antisense to transposon mRNAs
Argonaute3 complexes contain piRNAs biased to the sense strand of transposon mRNAs
piRNAs display 10 nt complementarity at their 5’-endsfrom Aravin et al., Science 318, 761 (2007)
Model for Biogenesis of piRNAs that Target Mobile Elements
Pool of piRNAs bound to Piwi or Aubergine anneals to transposon mRNA target
Cleave transposon mRNA 10 nt from 5’-end of associated piRNA to create 5’-end of Ago3 piRNA
Ago3-associated piRNA anneals to piRNA cluster transcript to create additional copies of antisense piRNA
Transposon is silenced
from Aravin et al., Science 318, 761 (2007)
from Marek, Nature 509, 570 (2014)
Role of piRNA in Sex Determination in Silkmoths
WZ – femaleZZ - male
piRNAs are transcribed from W chromosome in females and reduces Masc mRNA levels
Masc promotes male-specific splicing of doublesex
Sexual development is controlled by the sex-specific splicing of doublesex mRNA
Large ncRNAs
Much of the genome is transcribed
Many large ncRNAs contain modular domains that interact with chromatin regulators
Large ncRNAs can function as a molecular scaffold that forms a unique functional complex
Human genome encodes21,000 protein-coding genes9,000 small RNAs10,000 – 32,000 lncRNAs11,000 pseudogenes
from Wiedenheft et al., Nature 482, 331 (2012)
CRISPR is a Bacterial Defense Based on Small RNA
CRISPR contains repeats separated by unique spacers that arise from integration of short fragments of foreign DNA
CRISPR is a bacterial memory of past invasions
cas genes are linked to the CRISPR locus and are involved in integration, processing and interference
CRISPR RNA Biogenesis and Interference
from Wiedenheft et al., Nature 482, 331 (2012)
CRISPR loci are transcribed and processed into crRNAs
CRISPR RNA is processed by CRISPR-specific endonucleases or by RNaseIII cleavage of a tracrRNA-RNA duplex
crRNAs associated with Cas proteins, recognize and cleave foreign nucleic acids
Cas9 + crRNA + tracrRNA or (sgRNA) binds to PAM sites
Recognition of PAM promotes local unwinding and interrogates flanking DNA for the target
PAM binding activates the Cas9-RNA nuclease activity and generates a ds break
from Barrangou, Science 344, 707 (2014)
Cas9 Targeting and ds Break Formation
Specificity is determined by the crRNA sequence
Cas9 remains bound after cleavage to allow recruitment of DNA repair machinery
Self targeting is avoided since the CRISPR locus lacks PAMs