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Welcome Each of You to My Molecular Biology Class.

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1 Welcome Each of You to My Molecular Biology Class

2 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

3 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 4 Chapter 18 Regulatory RNAs Chapter 18 Regulatory RNAs Molecular Biology Course

5 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 6 PART 1 Regulation by RNAs in Bacteria. PART 2 RNA Interference and miRNA Regulation in Eukaryotes Chapter 18 Regulatory RNAs

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

8 8 1.Small RNAs (sRNA): regulation by base pairing. 2.Riboswitches: regulation by metabolite-mediated structure changes. 3.Attenuation: Regulation by ribosome stop-mediated formation of terminators

9 9 1.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] Figure 18-1

10 10 2. Riboswitches are 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 11 Figure The structure of a riboswitch in its regulated mRNA

12 12 代谢物 Figure 18-3a. Control of transcription termination by a riboswitch Figure 18-3a. Control of translation initiation by a riboswitch.

13 13 Figure Alteration of of the structure of the SAM riboswitch upon the binding of SAM (S-adenosyl methionine)

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

15 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 16 The trp operon encodes five structural genes required for tryptophan ( 色胺酸 ) synthesis. These genes are regulated to efficiently express only when tryptophan is limiting. (2) attenuation Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation The TRP operon

17 17 Fig 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 18 1. 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. 2. The leader peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome will pause at these sites. 3. 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 19 The leader RNA and leader peptide of the trp operon

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

21 21 Importance of attenuation 1. A typical negative feed-back regulation 2. Cellular tryptophan level is controlled by both repression and attenuation. 3. Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation. 4. The first example of gene regulation mediated by altering RNA secondary structure.

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

23 23 Outlines 1. RNAi discovery and mechanism 2. The discovery of miRNAs 3. miRNA biogenesis and regulation 4. miRNA roles in development, cell differentiation and virus 5. miRNA in cancer 6. siRNA application

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

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

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

27 27

28 28

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

30 30 Fig 3. Effects of mex-3 RNA interference on levels of the endogenous mRNA 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 ). No hybridization and staining + hybridization (endogenous mex-3 RNA) +antisense +hybridization +ds mex-3 RNA +hybridization

31 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 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) 33 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 34 Figure /18-6 RNAi silencing 外源双链 RNA siRNAs & miRNAs pre-miRNAs

35 35 1.Trigger destruction of the target mRNA ( 引起靶标 mRNA 的降解 ), 2.Inhibit translation of the target mRNA ( 抑制靶标 mRNA 的翻译 ), 3.Induce chromatin modification ( 引起靶 标启动子的转录沉默 ). Three ways of the RNAi-directed gene silencing (functional output)

36 36 Cleavage & degradation Multi-functional RNAi

37 37 The heart of the RNAi mechanism 1.Dicer: 1.Dicer: an RNaseIII-like multidomain ribonuclease that first processes input dsRNA into small fragments, siRNAs or miRNA. Dicer then helps load its small RNA products into RISC. 2.RISC(RNA induced silencing complexes) 2.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. RNAi machinery

38 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 39 the PAZ domain two catalytic RNase III domains 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 40 RISC: the key component is Argonaute (AGO) Argonaute (AGO) Argonaute (AGO) : A large protein family that constitutes key components of RISCs. two unique domains, PAZ and PIWIPAZ PIWI ---AGO proteins are characterized by two unique domains, PAZ and PIWI. PAZ domain 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 ---Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave the mRNA target, whereas AGO1 and AGO3 do not.

41 41

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

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

44 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 45 Transcriptional Gene Silencing by Directing Chromatin Modification RNAi output 3: Transcriptional Gene Silencing by Directing Chromatin Modification Figure Silencing of the centromere in S. pombe ( 裂殖酵母 )

46 46 RNA silencing in different organisms

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

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

49 49 生物的复杂性不由编码蛋白质 的数目决定 1. 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。 98 %的基因组有什 么功能 2. 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!】 人类基因组草图带给科学家们的困惑

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

51 51 The first discovered miRNA lin-4 in 1993 Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell Jan 23;116 (2 Suppl):S93-6. Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell Jan 23;116 (2 Suppl):S89-92 Thought to be an oddity not a general phenomenon

52 52 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 Nov 2;408(6808):86-9.

53 53

54 54 RNA 调控 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.

55 55 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。

56 56 microRNAs had been neglected for so many years because of their small size. The underlying reason is: people never dream that small RNAs will have important biological roles.

57 57 The number of the identified miRNAs is growing rapidly in recent years (now approaching late log phase)9539 has- miR The number of the identified miRNAs is growing rapidly in recent years (now approaching late log phase) miRNAs have been found until March, 2009 (The miRBase Sequence Database).These miRNAs are from primates, rodents, birds, fish, worms, flies, plants and viruses. Among which 706 miRNAs from human, which are named as has- miRx. The data are freely available to all through the web interface at and in flatfile form from

58 58 Topic 3: miRNA biogenesis and regulation 三、 miRNA 生成和调控 CHAPTER 18 RNAi and miRNA regulation

59 59 1. MicroRNA (miRNA) & its processing 微小 RNA 及其加工

60 60 MicroRNA (miRNA): 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 ).

61 61 The miRNA genes and Structure of pri-miRNAs Pri-miRNAs bear the 5’ cap and 3’ poly(A) tails

62 62 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

63 63 pri-miRNA 1. A typical metazoan pri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. the flanking ssRNA segments are critical for processing 2. The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. 3. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction.

64 64 Han et al., Cell 125, 887–901, June 2, 2006

65 65 Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain.

66 66 2. MicroRNA (miRNA) targeting and regulation.

67 67 Three strategies of miRNA and target recognition (targets are locating in 3’ UTRs).

68 68 A comparison between miRNA and siRNA

69 69 Topic 4: The biological roles of miRNAs (development, cell differentiation, virus-host interaction etc) CHAPTER 18 RNAi and miRNA regulation 四、 MicroRNA 广泛而重要的生物学功能

70 70 Victor R. Ambros 秀丽线虫 C. elegans 1. miRNA in C. elegans development

71 71 lin-4 and let-7 miRNAs lin-4 and let-7 miRNAs control the developmental timing of C. elegans.

72 72 Expression of lin-4 Expression of lin-4 allows C. elegans to proceed to the late developmental stage

73 73 lin-4binds its target mRNAs lin-4 binds its target mRNAs by imperfect base pairing.

74 74 2. miRNAs play important roles in vertebrate development.

75 75 Expression of miR-124a and miR-1 in Zebrafish, Medaka, Mouse, and Fly. Figure 2. Expression of miR-124a and miR-1 in Zebrafish, Medaka, Mouse, and Fly. miR-124a is restrictedly expressed in the brain and the spinal cord in fish and mouse or to the ventral nerve cord in the fly. The expression of miR-1 is restricted to the muscles and the heart in the mouse. 青鳉 斑马鱼 小鼠 果蝇 Learning the miRNA function from its expression pattern

76 76 3. miRNA controls plant phenotypes and more ( 控制植物表型特征 ) Jaw-miRNA 控制拟南芥叶形变化 (Nature, 2003)

77 77 ( Science 2004) 3 种 miRNA 控制造血干细胞向淋巴细胞的分化过程 4. miRNA controls the differentiation of the hematopoietic stem cell ( 调控造血干细 胞的分化 )

78 78 5. Some viruses encode miRNAs ( 有些病 毒编码 miRNAs)

79 79 Virus-related miRNA regulation

80 80 Topic 5: miRNA in cancer 五、微小 RNA 在癌症发生中的作用 CHAPTER 18 RNAi and miRNA regulation miRNA expression profile (phenomena): diagnosis markers, hits of biological roles miRNA regulatory mechanism miRNA roles in oncogenesis (biology and disease) Therapeutic application (forever topic)

81 81 miRNA expression pattern changes during oncogenesis, and is unique for each cancer. 微小 RNA 在癌症发生中表达谱的变化

82 82

83 83 Figure 3, Comparison between normal and tumor samples reveals global changes in miRNA expression.

84 84 One mechanism of miRNA controlling oncogene expression 微小 RNA 调控癌基因表达的一种机制。

85 85 1.c-Myc 1.c-Myc is a helix–loop–helix leucine zipper transcription factor that regulates an estimated 10–15% of genes in the human and Drosophila genomes. 2.c-Myc 2.c-Myc activates expression of a cluster of six miRNAs (mir cluster) on human chromosome 13. (Figure 1) 3.E2F1 3.E2F1 is the transcription factor, which is a target of c-Myc that promotes cell cycle progression. 4.Expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. 4.Expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR-17-5p and miR-20a. (Figure 4)

86 86

87 87 Used 2’-O-methyl Antisense oligonucleotides to downregulate the level of miR-17-5p and miR- 20a, and then analyzed the protein (B-Western) and mRNA levels (C-Northen) of E2F1.

88 88 Some microRNAs are potential oncogenes 有些微小 RNA 可能是致癌基因。

89 89 B- 细胞淋巴瘤

90 90 Figure 1. The mir-17–92 cluster shows increased expression in B-cell lymphoma samples and cell lines. Figure 1. The mir-17–92 cluster shows increased expression in B-cell lymphoma samples and cell lines. The level of mir-17–92 pri-miRNA was determined by real-time quantitative RT- PCR in 46 lymphomas and 47 colorectal carcinomas, and compared to levels found in corresponding normal tissues from five individuals.

91 91 Figure 2. Overexpression of the mir-17–19b cluster accelerates c-myc-induced lymphomagenesis in mice.

92 92 Topic 6: siRNA application CHAPTER 18 RNAi and miRNA regulation 六、 siRNA 的应用

93 93 1. siRNA application in mammalian Transfection of exogenous siRNA Chemical synthesis: fast, but expensive and short-term Generate esiRNA (enzymatic produced siRNA) from in vitro transcribed long dsRNA by E. coli RNase III or RNase III- like DICER: less expensive, large-scale, but short-term. Expression of siRNA siRNA produced with pol III promoter from the transfected DNA plasmids. Stable and long-term, but slower

94 94 1.Transcription from RNAP III promoters of U6 and H1 are well characterized. RNAP III transcription uses a well- defined termination signal (TTTTT) and the products have no extra sequence. 2. Transcription from these promoters is very efficient in various tissues. Expression of hairpin RNA (shRNA) using a Pol III promoter. 通过将编码 shRNA 的质粒转染进细胞内。 转染分两类:瞬时转染( transient transfection )和稳定转 染( Stable cell lines ) Expression of siRNA in cultured cells or in animal models

95 95 A mammalian expression vector designed to direct the intracellular synthesis of shRNAs, which will then be processed into siRNAs.

96 96 1. Create induced phenotypes that can be observed over long time spans. 2. Create a stably engineered cells can be assayed either in vitro ( 体外 - 细胞水平 ) or in vivo (体内), allowing the test of the angiogenic ( 血管生成 ) or metastatic ( 转移 ) potentials of tumor cells in xenograft models ( 异种移植模型 ) 。 3. Create hypomorphic alleles ( 亚等位基因 ) rapidly in transgenic mice. [Hypomorphic alleles are those in which function is reduced, but not completely lost] Advantages of expression of siRNA in living cells

97 97 4. Combine shRNAs with existing high- efficiency gene delivery vehicles to create bona fide RNAi-based therapeutics. For example, ultimately, to silence a disease-causing mutant allele specifically.

98 98 Research Applications of RNAi: A new strategy of reverse genetics & a novel way of gene knock-out It can be used in reverse genetics ( 反 向遗传学 ) to identify the cellular or biological function of a gene. It can be combined with genomics to perform large-scale genetic screens aimed at gene discovery.

99 99 Therapeutic Applications of RNAi: A new strategy to invitation of new drugs and gene therapy siRNAs can be used to counter viral infection by specifically destroying the mRNAs of the pathogenic viruses, such as HIV and HBV. siRNAs can be applied to counter cancers by specifically down-regulate the expression of genes related to oncogenesis.

100 100 本章重点掌握 1. 细菌中 RNA 调控的三种机制: sRNA, riboswitches, attenuation 2.RNA 干扰在真核基因表达调控中的功能 ( 1 ) RNA 干扰的发现和作用机制 ( 2 ) miRNA 的发现、生成机制和调控机 制。 ( 3 ) miRNA 的生物学功能,例子 lin-4 ( 4 ) miRNA 在癌症发生中的作用研究思 路 ( 5 ) siRNA 的产生方法及其在科研和治疗 上的应用。

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