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Homology-dependent Gene Silencing – The World in 1999 TGS – Pairing of tightly linked homologous loci induces methylation Transcriptional Gene Silencing PTGS – Transcript-specific degradation Post-transcriptional Gene Silencing SAS – Spread of PTGS Systemic Acquired Silencing RIP – Induction of C-T transitions Repeat-induced Point Mutation RNAi RNA interference from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 (1999) Small RNAs 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 tenOever, Nature Rev.Microbiol. 11, 169 (2013) Response of Mammalian Cells to Long dsRNA Long dsRNA induces interferon response in vertebrates PKR phosphorylates eIF2a 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 from McManus and Sharp, Nature Rev.Genet. 3, 737 (2002) 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 miRNAs Regulate Development in C. elegans 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 from Lodish et al., Molecular Cell Biology, 6th ed. Fig 21-6 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 ntlong 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) miRNA Biogenesis pri-miRNA is cleaved by Drosha at the base of a stem-loop structure pre-miRNA is exported from the nucleus by exportin 5 and cleaved by Dicer The duplex is loaded onto Argonaute protein miRNA* is expelled to produce the RISC from Ameres and Zamore, Nature Rev.Mol.Cell Biol. 14, 475 (2013) miRNA Target Recognition in Plants from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011) The 3’-end of plant miRNAs usually are modified with a 2’O-methyl group Plant miRNAs usually recognize fully complementary binding sites located in the ORF The PIWI domain of AGO cleaves the mRNA target between nucleotides 10-11 opposite the miRNA miRNA Target Recognition in Animals from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011) Animal miRNAs usually recognize partially complementary sites located in the 3’-UTR Complementarity within the seed region is the major determining of binding to the target Regulation of Small RNA Levels The presence of highly complementary target RNA triggers tailing and trimming In flies, small RNAs can be stabilized by methylation at the 3’-end from Ameres and Zamore, Nature Rev.Mol.Cell Biol. 14, 475 (2013) Triggers of RNAi-Mediated Gene Silencing in Mammals from Mittal, Nature Rev.Genet. 5, 355 (2004) Argonaute Proteins Argonaute proteins bind small RNAs and identify RNA targets by base-pairing Target silencing can occur by Target mRNA degradation Translation inhibition Recruitment of chromatin-modifying activities 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) Mechanisms of miRNA Sequence Diversification Differential processing results in miRNAs with different seed sequences In cells containing adenosine deaminase, A is converted to I from Ameres and Zamore, Nature Mol.Cell Biol. 14, 475 (2013) Causes of Off-Target Effects Sequence-based homology with non-target transcripts Aberrant processing or RNA editing General cell pertubations due to large amounts of RNA Incorporation of the passenger strand into the RISC 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) Mechanism of miRNA Action miRNAs Induce mRNA Degradation and Inhibit Translation miRNA-AGO binds to partially complementary sites in the target mRNA AGO recruits GW182 proteins from Izaurralde, Science 349, 380 (2015) miRNA-mediated Inhibition of Target mRNA Translation Translation repression accounts for a small part of target repression GW182 recruits deadenylase complexes to initiate mRNA degradation The miRISC interferes with the helicase function of eIF4A to inhibit translation initiation from Izaurralde, Science 349, 380 (2015) miRNA-mediated mRNA Degradation mRNA degradation is the dominant effect of miRNA-mediated regulation The decapping complex is recruited to the miRISC mRNA decapping makes the 5’-end accessible to the XRN1 nuclease from Izaurralde, Science 349, 380 (2015) Role of Poly(A) and Cap in Translation Initiation The cap structure is recognized by eIF4F Poly(A) is recognized by PABPC PABPC interacts with eIF4G from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011) Recruitment of the preinitiation complex is increased miRNAs Effects on Preinitiation Complex Formation miRISC-GW182 may compete with eIF4G for binding to PABPC and prevents mRNA circularization GW182 may reduce the affinity of PABPC for the poly(A) tail Preinitiation complex recruitment is inhibited from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011) Overview of RNA-Mediated Gene Silencing siRNA siRNA triggers endonucleolytic cleavage of perfectly-matched complementary targets Cleavage is catalyzed by Argonaute proteins The resulting mRNA fragments are degraded miRNA miRNA triggers accelerated deadenylation and decapping of partially-complementary targets and requires Argonaute proteins and a P-body component from Eulalio et al., Nature Rev.Mol.Cell Biol. 8, 9 (2007) miRNA degrades mRNA and represses translation Secretion of miRNAs Specific miRNAs can be preferentially sorted into vesicles and delivered to recipient cells from Chen et al., Trends Cell Biol. 22, 125 (2012) Tumor-derived Exosomes Initiate Tumor Growth in Normal Cells OncomiRs are incorporated into exosomes which are taken up by normal cells Exosomes may be used as biomarkers for cancer diagnosis Exosome mimetics containing miRNA sponges could fuse with normal cells and tumorderived exosomes to neutralize oncomiRs from Anastasiadou and Slack, Science 346, 1459 (2014) Regulation of siRNA Levels in C. elegans RNA-dependent RNA polymerase amplifies siRNA RRF-3 prevents siRNA amplification ERI-1 is an siRNA-specific RNase from Timmons, BioEssays 26, 715 (2004) Prevalence of and Regulation by miRNAs The human genome has the potential to encode >1500 miRNAs miRNAs control the expression of >50% of human proteins mRNAs can undergo negative selection to avoid a seed match miRNAs fine tune the expression of proteins in a cell Organismal Complexity May Be Due to Differences in Regulation of Gene Expression Number of protein-coding genes are similar in animals There is a continuous acquisition of novel miRNAs during evolution Lineage-specific loss of miRNAs also occurs miRNA complexity correlates with an increase in morphological complexity There are now estimated to be 1,424 miRNAs in humans from Technau, Nature 455, 1184 (2008) let-7 is a Heterochronic Gene in C. elegans 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 from Büssing et al., Trends Mol.Med. 14, 400 (2008) Regulation of Differentiation by let-7 let-7 levels are reduced in stem cells Lin28 promotes reprogramming by inhibition of let-7 maturation from Viswanathan and Daley, Cell 140, 445 (2010) Reprogramming to iPS Cells Oct4 Sox2 Klf4 c-Myc or Oct4 Sox2 NANOG Lin28 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 let-7 promotes differentiation 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 Droshamediated processing of let-7 During differentiation, let-7 targets Lin28 mRNA, which reinforces developmental commitment from Thornton and Gregory, Trends Cell Biol. 22, 474 (2012) Summary of Lin28 let-7 Regulation of Differentiation and Oncogenesis 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 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 neuronspecific 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 Down-regulation of all miRNAs enhanced tumor growth The undifferentiated state of malignant cells is correlated with a decrease in miRNA expression 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 from He et al., Nature 435, 828 (2005) Lu et al., Nature 435, 834 (2005) Lujambio and Lowe, Nature 482, 347 (2012) Inhibition of Endogenous miRNA function miRNA sponges Vectors express multiple copies of miRNA target sites Endogenous miRNA is saturated and prevented from silencing its natural product Pseudogene transcripts can act as miRNA sponges from Brown and Naldini, Nature Rev.Genet. 10, 578 (2009) Competitive Endogenous RNAs (ceRNAs) 70-90% of the human genome is transcribed, but less than 2% of the genome encodes protein-coding genes The human transcriptome contains 21.000 protein-coding genes, 9,000 small RNAs, 10,000-32,000 lncRNAs and 11,000 pseudogenes All RNA transcripts that contain miRNA binding sites that regulate each other by competing for shared miRNAs ceRNAs can fine-tune gene expression Regulation of PTEN Levels by a Pseuodogene 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 from Rigoutsos, Nature 465, 1016 (2010) PTENP1 RNA is a ceRNA that enhances PTEN expression by competing for a shared miRNA The PTEN ceRNA Network 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 from Tay et al., Nature 505, 344 (2014) 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 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 Kim and Rossi, Nature Rev.Genet. 8, 173 (2007) 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 Mittal, Nature Rev.Genet. 5, 355 (2004) Delivery of siRNA for Therapy from Dykxhoorn and Lieberman, Cell 126, 231 (2006) 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 Cell-Specific Delivery of siRNA 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 from Rossi et al., Nature Biotechnol. 23, 682 (2005) siRNA-mediated Pericentric Heterochromatin Formation in S. pombe Bidirectional transcription produces dsRNA that is processed into siRNA RNA-dependent RNA polymerase produces additional dsRNA and more siRNA is generated siRNA loaded onto Ago1 is guided to the nascent transcript from Castel and Martienssen, Nature Rev.Genet. 14, 100 (2013) Clr4 is recruited which methylates H3K9 3’-End Processing Prevents Transcriptional Silencing in S. pombe The effect of RNAi on chromatin is inconsistent Paf1C is essential for 3’-end processing of mRNA Paf1C prevents siRNA-mediated heterochromatin formation from Zaratiegui, Nature 520, 162 (2015) Small RNAs Modulate Viral Infection Viral-encoded miRNA facilitate viral infection and persistence Host cell-encoded miRNAs inhibit or facilitate viral replication Viral suppressors of RNA silencing (VSR) inhibit the RNAi pathway Function of SV40 miRNA 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 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 Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006) A MicroRNA was Thought to Protect HSV-1-infected Neurons from Apoptosis LAT is the only viral gene expressed during latent infection in neurons miR-LAT is generated from the LAT gene miR-LAT downregulates TGF-b and SMAD3 and contributes to the persistence of HSV-1 in neurons in a latent form from Gupta et al., Nature 442, 82 (2006) Paper retracted – 2008. Repeatedly unable to detect miRNA Cellular miRNAs Modulates Viral Infection PFV-1 replication is stimulated by a plant VSR implicating the role of small RNAs in the viral life cycle miR-32 inhibits viral replication Tas is a PFV-1-encoded protein that inhibits RNAi miR-122 increases HCV replication in the liver from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006) miR-122 stabilizes the HCV genome by binding the 5’-UTR miR-122 Protects the HCV Genome From Degradation 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 from Garcia-Sastre and Evans, Proc.Nat.Acad.Sci. 110, 1571 (2013) miRNA Encoded by an RNA Virus 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 from Cullen, Proc.Nat.Acad.Sci. 109, 2695 (2012) PIWI and piRNA The Drosophila PIWI phenotype – P-element-induced wimpy testis 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 Overview of piRNA Biogenesis in the Drosophila Female Germ Line Precursor piRNAs are transcribed from piRNA clusters Processing generates antisense piRNA containing a 5’-U piRNA is loaded onto AUB or PIWI and cleaves transposon RNA to produce sense piRNA containing 10A Additional antisense piRNA is produced by AGO3-mediated cleavage of piRNA precursors from Castel and Martienssen, Nature Rev.Genet. 14, 100 (2013) Precursor piRNA Production in Drosophila How are piRNA transcripts selectively processed into piRNAs? Rhino is an HP1 homolog that interacts with chromatin, UAP56, and precursor piRNAs UAP56 and Vasa are RNA helicases that facilitate transport of precursor piRNAs to their site of processing from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013) Primary piRNA Biogenesis in Drosophila ssRNA are precursors to piRNAs and production of piRNAs are Dicer-independent Zucchini may be the nuclease that cleaves piRNA precursors Piwi proteins may selectively bind RNAs that contain a 5’-U The 3’-end is trimmed and methylated to protect the piRNA from uridylation and degradation from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013) Secondary piRNA Biogenesis in Drosophila piRNA clusters often contain transposons Secondary piRNA processing ensures that the piRNA pool relates to the expression of their targets Aubergine bound to the primary piRNA cleaves the target RNA which is taken up by Ago3 The 3’-end is trimmed and methylated and binds to a precursor piRNA which is cleaved by Ago3 from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013) Role of piRNA in the Nucleus Piwi-piRNA can enter the nucleus and is responsible for H3K9 methylation to spread heterochromatin at transposon loci Transposon RNA is down-regulated from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013) Role of piRNA in Sex Determination in Silkmoths from Watanabe and Lin, Mol.Cell 56, 18 (2014) WZ – female ZZ - male Sexual development is controlled by the sex-specific splicing of doublesex mRNA Masc promotes male-specific splicing of doublesex piRNAs are transcribed from W chromosome in females and reduces Masc mRNA levels Large ncRNAs Much of the genome is transcribed Human genome encodes 21,000 protein-coding genes 9,000 small RNAs 10,000 – 32,000 lncRNAs 11,000 pseudogenes 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 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 cas genes are linked to the CRISPR locus and are involved in integration, processing and interference from Wiedenheft et al., Nature 482, 331 (2012) CRISPR is a bacterial memory of past invasions CRISPR RNA Biogenesis and Interference CRISPR loci are transcribed and processed into crRNAs CRISPR RNA is processed by CRISPRspecific endonucleases or by RNaseIII cleavage of a tracrRNA-RNA duplex crRNAs associated with Cas proteins, recognize and cleave foreign nucleic acids from Wiedenheft et al., Nature 482, 331 (2012) Cas9 Targeting and ds Break Formation 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 Specificity is determined by the crRNA sequence Cas9 remains bound after cleavage to allow recruitment of DNA repair machinery from Barrangou, Science 344, 707 (2014) Self targeting is avoided since the CRISPR locus lacks PAMs The Type II CRISPR System is Used for Targeted Genome Editing sgRNA generates a predictable ds break adjacent to a PAM site Break is repaired by NHEJ to knockout gene Presence of a homologous template allows genome editing using HR from Barrangou and Marraffini, Mol.Cell 54, 234 (2014) sgRNA-Guided Transcription Repression A catalytically dead Cas9 and sgRNA is targeted to any genomic location Targeting to a promoter represses transcription initiation from Barrangou and Marraffini, Mol.Cell 54, 234 (2014) Targeting to the template strand represses transcription elongation dCas9 Targeting of Enzymatic Activities to Specific Genomic Locations dCas9 can be fused to any functional domain Chimeric protein can be targeted to any genomic location by sgRNA from Barrangou and Marraffini, Mol.Cell 54, 234 (2014) Avoiding Off-target Effects in Genome Editing Cas9 is converted to a nickase Two sgRNAs directs nicks on opposite strands at adjacent sites from Ran et al., Cell 154, 1380 (2013) CRISPR-Cas9 Targeting of ssRNA from O’Connell et al., Nature 516, 263 (2014) A PAMmer anneals upstream of the target sequence A 5’-extended PAMmer is required for CRISPR-CAS9 cleavage of ssRNA