Understanding Homology-dependent Gene Silencing Pathways

1. 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)

2. Small RNAs from tenOever, Nature Rev.Microbiol. 11, 169 (2013)

3. 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)

4. 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)

5. 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)

6. 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

7. 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

8. 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)

9. 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)

10. 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

11. 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 determinant of binding to the target

12. 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)

13. Triggers of RNAi-Mediated Gene Silencing in Mammals from Mittal, Nature Rev.Genet. 5, 355 (2004)

14. 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

15. 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)

16. 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)

17. 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

18. 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)

19. Mechanism of miRNA Action

20. miRNAs Induce mRNA Degradation and Inhibit Translation miRNA-AGO binds to partially complementary sites in the target mRNA AGO recruits GW182 proteins from Izaurralde, Science349, 380 (2015)

21. 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, Science349, 380 (2015)

22. 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, Science349, 380 (2015)

23. 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 Recruitment of the preinitiation complex is increased from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)

24. 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)

25. 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 miRNA degrades mRNA and represses translation from Eulalio et al., Nature Rev.Mol.Cell Biol.8, 9 (2007)

26. 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)

27. 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 tumor- derived exosomes to neutralize oncomiRs from Anastasiadou and Slack, Science346, 1459 (2014)

28. 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, BioEssays26, 715 (2004)

29. 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

30. 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, Nature455, 1184 (2008)

31. 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)

32. 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, Cell140, 445 (2010)

33. Reprogramming to iPS Cells Oct4 Sox2 Klf4 c-Myc Oct4 Sox2 NANOG Lin28 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

34. 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

35. 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 Drosha- mediated 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)

36. 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

37. 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., Nature435, 828 (2005) Lu et al., Nature435, 834 (2005) Lujambio and Lowe, Nature482, 347 (2012)

38. miRNA Sponges Inhibit Endogenous miRNA Function from Thomson and Dinger, Nature Rev.Genet. 17, 272 (2016) Pseudogene transcripts, mRNAs, viral RNAs, circRNAs, and lncRNAs can compete for miRNAs Endogenous miRNA is saturated and prevented from silencing its natural product Physiological levels of ceRNA expression do not affect the function of highly expressed miRNAs

39. 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

40. 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 PTENP1 RNA is a ceRNA that enhances PTEN expression by competing for a shared miRNA from Rigoutsos, Nature465, 1016 (2010)

41. 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., Nature505, 344 (2014)

42. 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, Science340, 440 (2013)

43. 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)

44. 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)

45. Delivery of siRNA for Therapy from Dykxhoorn and Lieberman, Cell126, 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

46. 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)

47. 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

48. 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, Nature520, 162 (2015)

49. 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

50. Function of SV40 miRNA SV40 miRNA is synthesized late in the viral life cycle and targets TAg mRNA SV40 miRNA aids immune evasion by reducing susceptibility to lysis by CTLs from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006)