Biogenesis and the r egulation of Small RNAs

1. Biogenesis and theregulation of Small RNAs Genomics Evren Coşkun Becit

2. Guidelines • Types of sRNAs and sRNA Biogenesis • sRNA driven regulatory mechanisms • Conclusion • Future perspectives

3. Biogenesis of miRNA • Transcribed from MIR gene transcripts and are cleaved from stem-loop hairpins formed from RNA polymerase II transcripts. • miRNAs are processed via the action of the RNAse III enzymes (Drosha and Dicer in animals, DCLin plants) • After cropping and exportation to the cytoplasm, miRNAs are further diced and are loaded into the Argonaute proteins. • Drosha–Di George syndrome critical region gene 8 (DGCR8) acts as a ruler to measure the cleavage site. • Canonical intronic miRNAs are processed co-transcriptionally before splicing. • Non-canonical intronic small RNAs are produced from spliced introns and debranching. 7-methylguanosine Biogenesis of small RNAs in animals.Kim VN, Han J, Siomi MC. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39. miRNa biogenesis pathways in human and flies

4. Mechanisms of miRNA-mediated gene regulation • Following Dicer cleavage, the resulting ~22‑nt RNA duplex is loaded onto an Ago protein. The guide strandremains as a mature miRNA whereas the passenger strand is cleaved by AGo and degraded. • The strand with relatively unstable (thermodynamically) base pairs at the 5′end typically survives. • AGo-miRNA then incorporates into RISC. miRISC binds to target mRNA at 3`UTR which leads to translation repression of target genes. The imperfect base-pairing, resulting in a bulge structure, avoids AGo2 cleavage. • Translation-repressed mRNA and miRISC are concentrated in P-bodies for storage or mRNA decay. • Stored mRNAs can be released and re-enter translation pathways. Small RNAs: regulators and guardians of the genome. Chu CY, Rana TM. J Cell Physiol. 2007 Nov;213(2):412-9.

5. miRNA target recognition • Slicing-dependent reduction in mRNA accumulation, and/or translational repression. • Central bulge results in active AGo-miRNA complexes. • Seed pairing in animal differes from cannonical plant pairing in size. Classification and comparison of small RNAs from plants. Axtell MJ. Annu Rev Plant Biol. 2013;64:137-59.

6. miRNA mediated translation repression • Post-initiation repression mechanism. • Initiation-repression mechanism:Binding of AGo to m7G-cap prevents the recruitment of eIF4E(translation initiation factor). miRISC recruits elF6 and elF6 blocks40s subunit association which in turn hinders the 80s assembly. Thereby translation initiation is blocked. • Destabilisation of mRNA:mRNArecognition by miRNA destabilizes the target by inducing its deadenylation and decay. Small RNAs: regulators and guardians of the genome. Chu CY, Rana TM. J Cell Physiol. 2007 Nov;213(2):412-9.

7. Biogenesis of piRNA • Primary precursor piRNAs are transcribed fromintergenetic repetitive elements, active transposon genes and piRNA clusters.It is postulated that piRNAs use nuclease activity of the Piwi proteins for their processing. • piRNA biogenesis involves primary and secondary processing mechanisms. Primary processing is not well defined. • Primary processing generates antisense piRNAs which have a 5ʹ uridine (5ʹ U). • Sense transposon mRNA is cleaved to produce sense piRNAs, which have a strong adenine bias at position 10 (10A). • Antisense piRNA is produced by AGO3‑mediated cleavage of antisense primary piRNA transcripts. • Only loaded PIWI is imported into the nucleus. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.

8. piRNA Transposon silencing • piRNAs silence the TEs through the cleavage of TE-derived transcripts or DNA methylation of the genomic loci to cause transcriptional silencing. • In follicle cells flamenco cluster generates piRNAs. • In oocytes and surrounding nurse cells, Active transposons are post-transcriptionally silenced and nuclear PIWI promotes transcriptional silencing by methylation ofhistone H3 at lysine 9 (H3K9me) and heterochromatin protein 1A (HP1A) localization. • HP1A homologue Rhino binds to heterochromatic piRNA clusters in place of HP1A and promotes transcription. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.

9. Biogenesis of siRNA • Exogenous small interfering RNAs are derived from double-stranded dsRNAs or viral RNAs. • Endogenous siRNA (endo-siRNA) precursors are derived from repetitive sequences, sense–antisense pairs or long stem-loop structures. • siRNA precursors are transferred to cytoplasm and are loaded onto Dicer-TRBP complex to generate 21- to 22-nt siRNA duplexes. • siRNA duplex is loaded onto AGo while the passenger strand is removed. • Active siRISCrecognizes and cleaves its target mRNA. Exo- and Endo-sirNa biogenesis pathways in human Biogenesis of small RNAs in animals.Kim VN, Han J, Siomi MC. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.

10. siRNA mediated transposon silencing • In male germ line transposon transcripts are processed into 21 nt siRNAs and mobile siRNAs direct post-transcriptional gene silencing (PTGS) in the sperm nuclei. • In female gametophyte DME1 expression and MET1 repression yields to Transposon activation. This activates the RNA-directed DNA methylation (RdDM) pathway and produces 24 nt siRNAs. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.

11. siRNA-directed DNA methylation • RNA-DEPENDENT POLYMERASE 2 (RDR2) physically associates with RNA Pol IV to produce dsRNA. • In the cytoplasm siRNA is facilitated by HSP90, and the loaded AGO4is then imported back into the nucleus. • AGO4 targets nascent RNA Pol V transcripts and forms the RNA-directed DNA methylation (RdDM) complex. • PolV associated protein KTF1 interacts with AGO 4 and 5-methylcytosine. • Interaction between DRM2 and AGO4 (via RDM1) creates a positive-feedback loop between AGO4 localization and DNA methylation. • After it has been localized, DRM2 catalyses methylation of cytosine in all sequence contexts. The RNA-dependent DNA methylation pathway in Arabidopsis thaliana RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.

12. miRNAs with imperfect complementarity to their targets cause translational repression. piRNAs which target transposon transcripts in animal germ lines. siRNAs which have perfect complementarity to targets and cause transcript degradation and effect heterochromation formation. The miRNA biogenesis pathway is well studied in comparison to piRNA and siRNA pathways, yet many questions remain still unanswered. Conclusion Biogenesis of small RNAs in animals.Kim VN, Han J, Siomi MC. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.

13. Future perspectives • Since siRNAs can be employed as a biological tool to knock down specific genes, the most important issue in designing siRNAs is how to increase their potency and ensure specific gene silencing. • New mechanisms of gene regulation and genome stability as well as to enhance the potential for new RNAi-based therapies. • Understanding the underlying mechanisms that effect the small RNA stability will benefit the design and utilization of small RNAs for genetic manipulations.

14. References • Kawaji H1, Nakamura M, Takahashi Y, Sandelin A, Katayama S, Fukuda S, Daub CO, Kai C, Kawai J, Yasuda J, Carninci P,Hayashizaki Y. Hidden layers of human small RNAs. BMC Genomics. 2008 Apr 10;9:157. • Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39. • Daniel Klevebring, Nathaniel R Street, Noah Fahlgren, Kristin D Kasschau, James C Carrington, Joakim Lundeberg, Stefan Jansson. Genome-wide profiling of Populus small RNAs. BMC Genomics. 2009; 10: 620.  • Vaughn MW, Martienssen R. It's a small RNA world, after all. Science. 2005 Sep 2;309(5740):1525-6. • Ji L, Chen X. Regulation of small RNA stability: methylation and beyond. Cell Res. 2012 Apr;22(4):624-36. • Castel SE, Martienssen RA. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat Rev Genet. 2013 Feb;14(2):100-12. • Billi AC, Fischer SE, Kim JK. Endogenous RNAi pathways in C. elegans.WormBook. 2014 May 7:1-49. • Ba Z, Qi Y. Small RNAs: emerging key players in DNA double-strand break repair. Sci China Life Sci. 2013 Oct;56(10):933-6. • Chu CY, Rana TM. Small RNAs: regulators and guardians of the genome. J Cell Physiol. 2007 Nov;213(2):412-9. • Axtell MJ. Classification and comparison of small RNAs from plants.Annu Rev Plant Biol. 2013;64:137-59. • Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009 Feb;10(2):126-39.

15. Thankyouforyourpatience

16. Additional information Regulation of small RNA stability: methylation and beyond. Ji L, Chen X. Cell Res. 2012 Apr;22(4):624-36. Epub 2012 Mar 13.

17. Factors effecting sRNA stability Regulation of small RNA stability: methylation and beyond. Ji L, Chen X. Cell Res. 2012 Apr;22(4):624-36. Epub 2012 Mar 13.

18. 3′ uridylation generally marks small RNAs for degradation and reveal other 3′ tailing events that influence small RNA stability. Regulation of small RNA stability: methylation and beyond. Ji L, Chen X. Cell Res. 2012 Apr;22(4):624-36. Epub 2012 Mar 13.

19. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Castel SE, Martienssen RA. Nat Rev Genet. 2013 Feb;14(2):100-12.