Chapter 7: RNAi and miRNA regulation

1. Chapter 7: RNAi and miRNA regulation

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

3. 一、miRNA发现的背景和miRNA发现 Topic 1: The background and discovery of miRNAs

4. 后基因组时代的基因调控：RNA 调控 Most of the RNA transcribed from your genome doesn’t make protein. Carina Dennis talks to the revolutionaries who believe that it functions in gene-regulatory networks that underlie the complexity of higher organisms.

5. 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？当然，在这98％的非蛋白质编码基因组序列里，约24％为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？

6. 人类基因组绝大部分都被转录成RNA，细胞内非编码RNA的数量是编码RNA的上百倍。这促使许多科学家认为生物体复杂性被隐藏在它们所输出的非编码RNA内，而非编码序列内。人类基因组绝大部分都被转录成RNA，细胞内非编码RNA的数量是编码RNA的上百倍。这促使许多科学家认为生物体复杂性被隐藏在它们所输出的非编码RNA内，而非编码序列内。

7. 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)

8. RNAi研究——Hot topics in the world • 2000年，RNAi的研究进展被Science杂志评为重大科技突破； • 2001年“RNAi”作为当年最重要的科学研究成果之一，再次入选“十大科技突破”； • 2002年12月20日，Science杂志将“Small RNA & RNAi”评为2002年度最耀眼的明星。同时，Nature杂志亦将Small RNA评为年度重大科技成功之一。 • 2003年，microRNA研究第四次入选“十大科技突破”，排在第四位。 • 2005年，microRNA研究第五次入选“十大科技突破”。 • RNA研究的突破性进展，是生物医学领域近20年来，可与HGP相提并论的最重大成果之一。

9. The first discoered miRNA: lin-4 Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell. 2004 Jan 23;116 (2 Suppl):S93-6. Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell. 2004 Jan 23;116 (2 Suppl):S89-92 Thought to be an oddity not a general phenomenon

10. Breakthrough with BlastN of the second miRNA (stRNA) let-7 Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Muller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P,Davidson E, Ruvkun G. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000 Nov 2;408(6808):86-9.

11. microRNAs had been neglected for so many years because of their small size. OOPs!

13. The number of the identified miRNAs is growing rapidly in recent years. Over >10000 miRNAs have been found until the August of this year (The miRBase Sequence Database). These miRNAs are from primates, rodents, birds, fish, worms, flies, plants and viruses. The data are freely available to all through the web interface at http://microrna.sanger.ac.uk/sequences/ and in flatfile form from ftp://ftp.sanger.ac.uk/pub/mirbase/sequences/.

15. Mature miRNA • 21-25nt ssRNA • Highly conserved • A more restricted spatial and temporal expression pattern • Slightly regulation • About 1 thousand of human miRNAs regulate 1/3 human genes

16. Homology Between C. elegans and Homo sapiens miRNAs

17. Predicted miRNA Precursors

19. 二、RNA干扰及其机制 Topic 2: RNA interference and its mechanism

20. 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

21. 2006年的诺贝尔生理学奖获得者： Andrew Z. Fire Craig C. Mello

22. 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)

23. The discovery of RNAi explains the virus-induced gene silencing in plants (植物病毒引起的基因沉默). 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.

24. 2. Short interfering RNA (siRNAs) are produced from dsRNA and direct machinery that switch off genes in various way. [Mechanism-机制] 从双链RNA产生的小干扰RNA可以指导用不同机制关闭基因的细胞机器

25. Terms • RNAi: RNA interference. Degradation of mRNA molecules specified by complementary double-stranded RNA • dsRNA: double-stranded RNA. Complementary RNA molecules which form a double helix • siRNA: small interfering RNA: RNA duplexes of 21-23 nucleotide pairs with 2 unpaired nucleotides at each 3’ end. Identify mRNA molecules for degradation in RNAi. • RISC: RNA-induced silencing complex. Including proteins and siRNAs and degrades mRNA molecules • PTGS: posttranscriptional gene silencing: A means of gene regulation in plants which occurs after the production of mRNA. • PPD: PAX and PIWI domain. Proteins, including those involved in RNAi, PTGS and quelling, that contain the above two protein domains.

27. RNA Interference • Post-transcriptional gene silencing • First discovered in c. elegans and plants • Protective role: parasitic and viral resistance • Mammals • RNAi occurs –

28. Mechanism of siRNA RNAi 2 step mechanistic model: • Initiation step • Generation of siRNAs • Effector step • Degradation of target mRNA

29. Initiation Step ATP DICER ADP + ppi ATP KINASE ADP + ppi Effector Step • siRNA binding • siRNA unwinding • RISC activation

30. Mechanism of RNAi Processing the dsRNA precursors When dsRNAs are processed by Dicer, RNA duplexes with a length of about 21 nt are produced. RNA silencing effector complex assembly The siRNAs are subsequently incorporated into a multiprotein complex. This complex is known as the RNA-induced silencing complex (RISC). mRNA cleavage and repression of translation Once the RISC complex has been formed, the siRNAs in the RISC complex guide degradation that is sequence-specific, of the complementary or near complementary mRNAs. The RISC works by cleaving the mRNA in the middle of its complementary region.

31. Dicer • Family of RNase III enzymes • Recognize and process dsRNA into siRNA • Each dicer enzyme has an amino terminal helicase domain, 2 RNase III catalytic domains, dsRNA binding domain and a PAZ domain • Dicer is thought to act as a dimer of 2 enzymes

32. The question to be addressed is “Why exogenous dsRNA can inhibit expression of genes homologous to that RNA?”

33. Exogenous dsRNA 外源双链RNA

34. The targets of the RNAi-directed gene silencing • Degradation of the target mRNA (引起靶标mRNA的降解), • Inhibition of translation of the target mRNA (抑制靶标mRNA的翻译), • Silencing the gene transcription from the target promoter (引起靶标启动子的转录沉默).

35. The heart of the RNAi mechanism • Dicer: an RNaseIII-like multidomain ribonuclease that first processes input dsRNA into small fragments called short interfering RNAs (siRNAs) or microRNAs (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.

36. 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

37. 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.

38. 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, whose functions are not fully understood. Current evidence suggests that the PAZ domain binds the 3’-end two-nucleotide overhangs of the siRNA duplex, whereas the PIWI domain of some AGO proteins confers slicer activity. PAZ and PIWI domains are both essential to guide the interaction between the siRNA and the target mRNA for cleavage or translational repression. ---Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave the mRNA target, whereas AGO1 and AGO3 do not.

40. A model for siRNA-guidedmRNA cleavage by Argonaute

41. The multiple functions of RNAi

42. 三、miRNA生成和调控 Topic 3: miRNA biogenesis and regulation

43. 1. MicroRNA (miRNA) & its processing 微小RNA及其加工

44. MicroRNA (miRNA):A type of non-coding small RNA (~21–23 nucleotides) produced by Dicer from a stem-loop structured RNA precursor (~70-90 nts ong) (结构和来源). miRNAs are widely expressed in animal and plant cells and functions in the form of RNA–protein complexes, termed miRISCs. miRNAs have been implicated in the control of development because they lead to the destruction or translational suppression of target mRNAs with homology to the miRNA (生物学功能和机制).

45. The miRNA genes and Structure of pri-miRNAs Pri-miRNAs bear the 5’ cap and 3’ poly(A) tails

46. miRNA processing Pri-miRNA (miRNA初级转录产物) Drosha (1) pre-miRNA (miRNA前体) Dicer (2) miRNA Exportin 5 (Exp5) transports pre-miRNA to the cytoplasm

47. A typical metazoan pri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. • The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. • The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction.

48. Han et al., Cell 125, 887–901, June 2, 2006

49. Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain.

50. 2. MicroRNA (miRNA) targets and regulation.