Welcome Each of You to My Molecular Biology Class

1. Welcome Each of You to My Molecular Biology Class

2. Molecular Biology of the Gene, 5/E--- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 2005-5-10

3. Part IV Regulation Ch 16: Transcriptional regulation in prokaryotes Ch 17: Transcriptional regulation in eukaryotes Ch18: Regulatory RNAs Ch 19: Gene regulation in development and evolution Ch 20: Genome Analysis and Systems Biology

4. Chapter 18 Regulatory RNAs • Molecular Biology Course

5. TOPIC 1 Regulation by RNAs in Bacteria. TOPIC 2 RNA Interference Is a Major Regulatory Mechanism in Eukaryotes. TOPIC 3 Synthesis and function of miRNA molecules. TOPIC 4 The Evolution and Exploitation of RNAi. TOPIC 5 Regulatory RNAs and X-inactivation.

6. Chapter 18 Regulatory RNAs PART 1 Regulation by RNAs in Bacteria. PART 2 RNA Interference and miRNA Regulation in Eukaryotes

7. Chapter 18 Regulatory RNAs PART 1 Regulation by RNAs in Bacteria

8. Small RNAs (sRNA): regulation by base pairing. Riboswitches: regulation by metabolite-mediated structure changes. Attenuation: Regulation by ribosome stop-mediated formation of terminators

9. Small RNAs (sRNA) ---Regulation of translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively. [Targets] ---Regulation by base pairing with the targeted sequences on mRNAs. [Mechanism] ---Acts in trans similar to miRNA, but does not require specific machinery for action. [Mechanism] Figure18-1

10. 2. Riboswitchesare regulatory RNA elements that act as direct sensors of small molecule metabolites to control gene transcription or translation. ---Regulate translation initiation and transcription termination by altering the accessibility of RBS and the formation of terminator, respectively. [Targets] ---Reside upstream of the targeted mRNA, and form specific structure to bind its small molecule ligand. [Mechanism] ---Act in cis by alteration of its own structure upon the binding of the small metabolites. [Mechanism]

11. Figure18-2. The structure of a riboswitch in its regulated mRNA

12. Figure18-3a. Control of transcription termination by a riboswitch 代谢物 代谢物 Figure18-3a. Control of translation initiation by a riboswitch.

13. Figure18-4. Alteration of of the structure of the SAM riboswitch upon the binding of SAM (S-adenosylmethionine)

14. The 2nd structures of 7 riboswitches and metabolites that they sense

15. RNA Regulation in Bacteria 3. Attenuation (衰减作用) ---A premature transcription termination that switches off gene expression from amino acid biosynthetic operons after the corresponding amino acid is synthesized at an adequate level. [target] ---Requires the participation of ribosomes that translate a leader peptide. The premature transcription termination is triggered by formation of an intrinsic terminator when ribosome read through codons of the amino acid that the operon synthesizes. [mechanism].

16. The TRPoperon • The trpoperon encodes five structural genes required for tryptophan (色胺酸) synthesis. • These genes are regulated to efficiently express only when tryptophan is limiting. • Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation

17. Fig 16-19 Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough, which results in the production of a leader RNA of 161 nt. (WHY?)

18. Transcription and translation in bacteria are coupled (细菌体内的转录和翻译是偶联的).Therefore, synthesis of the leader peptide immediately follows the transcription of leader RNA. The leader peptidecontains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites. Ribosome pause at these sites alter the secondary structure of the leader RNA, which eliminates the intrinsic terminator structure and allow the successful transcription of the trp operon.

19. The leader RNA and leader peptide of the trp operon

20. High Trp RNA Pol Transcription of the leader RNA. Low Trp RNA Pol Transcription of the trp operon mRNA.

21. Importance ofattenuation A typical negative feed-back regulation Cellular tryptophan level is controlled by both repression and attenuation. Attenuation alone can provide robust regulation: other amino acids operons like his and leuhave no repressors and rely entirely on attenuation for their regulation. The first example of gene regulation mediated by altering RNA secondary structure.

22. Chapter 18 Regulatory RNAs PART 2 RNAi and miRNA regulation

23. Outlines RNAi discovery and mechanism The discovery of miRNAs miRNA biogenesis and regulation miRNA roles in development, cell differentiation and virus miRNA in cancer siRNA application

24. CHAPTER 18 RNAi and miRNA regulation Topic 1: RNA interference is a major regulatory mechanism in eukaryotes

25. 1 Double-stranded RNA inhibits expression of genes homologous to that RNA. [phenomena-现象] 双链RNA抑制含其同源序列基因的表达

26. 2006年的诺贝尔生理学奖获得者： Andrew Z. Fire Craig C. Mello 发现于1998

29. Fig 2. Analysis of RNA-interference effects in individual cells. Fluorescence micrographs show progeny of injected animals from GFP-reporter strain PD4251 (a C. elegans strain expressing GFP fluorescence protein) (使用外源导入的报告基因). ds-gfp RNA Control dsRNA Young larva (幼虫) Adult (成虫) adult body wall at high magnification (高放大倍数的 成虫体壁)

30. Fig 3. Effects of mex-3 RNA interference on levels of the endogenous mRNA (in situ hybridization in embryos) (胚胎的原位杂交). + hybridization (endogenous mex-3 RNA) No hybridization and staining +ds mex-3 RNA +hybridization +antisense +hybridization

31. Importance of RNAi discovery: explains the virus-induced gene silencing in plants (植物病毒引起的基因沉默) found years ago. Most plant viruses have single-stranded RNA genomes, which are released from the protein coat of their virus particles as they enter a cell. Their genomic RNA is then replicated by the virus encoded RNA-dependent RNA polymerase to produce sense and antisense RNA, which can hybridize to form dsRNA and trigger an RNAi response against their own sequences.

32. 2. Short RNAs that silence genes are produced from a variety of sources and direct the silencing of genes in three different ways [机制]

33. Short RNAs (functional input) Different small silencing RNAs are named according to their origin ---siRNAs (small interfering RNAs): made artificially or produced in vivo from dsRNA precursors ---miRNA (microRNA): derived from precursor RNAs encoded by nuclear genes.

34. 外源双链RNA siRNAs & miRNAs pre-miRNAs Figure 17-30 /18-6 RNAi silencing

35. Three ways of the RNAi-directed gene silencing (functional output) • Trigger destruction of the target mRNA (引起靶标mRNA的降解), • Inhibit translation of the target mRNA (抑制靶标mRNA的翻译), • Induce chromatin modification (引起靶标启动子的转录沉默).

36. Multi-functional RNAi Cleavage & degradation

37. RNAi machinery • The heart of the RNAi mechanism • Dicer: an RNaseIII-like multidomainribonuclease that first processes input dsRNA into small fragments, siRNAs or miRNA. Dicer then helps load its small RNA products into RISC. • RISC(RNA induced silencing complexes) (RNA诱导的沉默复合体): a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.

38. Dicer: Structural organization: ---A PAZ domain, binds the end of the dsRNA ---Two RNase III domains ---Other non-conserved domains. 贾第鞭毛虫

39. The crystal structure of the Giardia intact Dicer enzyme shows that the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic RNase III domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.

40. RISC: the key component is Argonaute (AGO) Argonaute (AGO): A large protein family that constitutes key components of RISCs. ---AGO proteins are characterized by two unique domains, PAZ and PIWI. PAZdomain binds the 3’ end 2 nt overhangs of the guide strand of siRNA/miRNA, whereas the PIWI domain (RNase III) confers slicer activity. The cleavage occurs in the middle of guide RNA-target RNA duplex. PAZ and PIWI domains are both essential for Ago activity. ---Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave the mRNA target, whereas AGO1 and AGO3 do not.

42. RNAi output 1:A model for siRNA-guided mRNA cleavage by Ago

43. RNAi output 2:Mechanisms of miRNA-Mediated Translational Inhibition Eulalio et al. Cell 132, 2008

44. (A) Inhibition of translation elongation: miRNAs repress translation of target mRNAs by blocking translation elongation or by promoting premature dissociation of ribosomes (ribosome drop-off) (B) Co-translational protein degradation: The protein is normally translated after which it is immediately degraded proteolytically. (C) Competition for the cap structure: Argonaute proteins compete with eIF4E for binding to the cap structure. (D) Inhibition of ribosomal subunit joining: Argonaute proteins recruit eIF6, which prevents the large ribosomal subunit from joining the small subunit.

45. RNAi output 3:Transcriptional Gene Silencing by Directing Chromatin Modification Figure 18-13. Silencing of the centromere in S. pombe (裂殖酵母)

46. RNA silencing in different organisms

47. CHAPTER 18 RNAi and miRNA regulation 一、miRNA发现的背景和miRNA发现 Topic 2: miRNA discovery

48. Central dogma in post-genomic era: gene regulation 基因组的保持 基因组的表达 RNA processing Gene regulation

49. 人类基因组草图带给科学家们的困惑 • 含有30亿对碱基的人类基因组仅含有2－3万个蛋白质基因，是果蝇的两倍，啤酒酵母的4倍。显而易见，生物的复杂性不由编码蛋白质的数目决定。 • 人类基因组的蛋白质编码区的总和占总基因组长度为1－2％，那么其他98％的基因组有什么功能呢？(1) 24％的基因组是插入编码序列的内含子序列；人类基因平均每个基因有7个内含子。但这么冗长的内含子序列有什么生物学功能呢？ (2) 其他74%的基因组的功能是什么？【注：90%以上的基因组都是转录的！】

50. The discovery of miRNAs Victor Ambros Gary Ruvkun • miRNA was first discovered in 1993 by Victor Ambros at Harvard (lin-4) • The second miRNALet-7 was discovered in 2000 by Frank Slack as a postdoc at Harvard(Ruvkun lab)