分子生物学教学课件：Part 4 chapter18 Regulatory RNAs

1、1Part IV RegulationCh 16: Transcriptional regulation in prokaryotesCh 17: Transcriptional regulation in eukaryotesCh18: Regulatory RNAsCh 19: Gene regulation in development and evolutionCh 20: Genome Analysis and Systems Biology2Chapter 18Regulatory RNAsMolecular Biology Course3TOPIC 1: Regulation b

2、y 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.4Chapter 18 Regulatory RNAsPART 1:Regulation by RNAs in Bacteria5S

3、mall RNAs (sRNA): regulation by base pairing.Riboswitches: regulation by metabolite-mediated structure changes.Attenuation: Regulation by ribosome stop-mediated formation of terminators 6Small RNAs (sRNA) 82. Riboswitches（核酸开关） are regulatory RNA elements that act as direct sensors of small molecule

4、 metabolites to control gene transcription or translation.9代谢物代谢物Figure 18-3b. Control of transcription termination by a riboswitchFigure 18-3a. Control of translation initiation by a riboswitch.1011The 2nd structures of 7 riboswitches and metabolites that they sense 12RNA Regulation in Bacteria3. A

5、ttenuation (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 transcr

6、iption termination is triggered by formation of an intrinsic terminator when ribosome read through codons of the amino acid that the operon synthesizes. mechanism.13The trp operon encodes five structural genes required for tryptophan (色胺酸) synthesis.These genes are regulated to efficiently express o

7、nly when tryptophan is limiting.Two layers of regulation are involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuationThe TRP operon14Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough, which results in the production of

8、a leader RNA of 161 nt. (WHY?)15Transcription 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 peptide contains two tryptophan codons. If the tryptophan level is very low, the ribosome

9、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. 1617Low TrpHigh TrpTranscription of the leader RNA.Transcription of the trp operon mR

10、NA.RNA PolRNA Pol18Importance of attenuation A typical negative feed-back regulationCellular tryptophan level is controlled by both repression and attenuation.Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuati

11、on for their regulation.The first example of gene regulation mediated by altering RNA secondary structure.19Chapter 18 Regulatory RNAsPART 2 RNAi and miRNA regulation20OutlinesRNAi discovery and mechanismThe discovery of miRNAsmiRNA biogenesis and regulationmiRNA roles in development, cell different

12、iation and virusmiRNA in cancersiRNA application21CHAPTER 18 RNAi and miRNA regulationTopic 1: RNA interference is a major regulatory mechanism in eukaryotesBCB 444/544 F08 ISU Terribilini 34 - miRNA22Timeline for miRNA DiscoveriesEntrez PubMed Search Terms: RNAi or siRNA or miRNANumber of Publicati

13、ons241. 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Early demonstration of RNAi in plantsIn situ hybridization for mex-3 mRNA 4 cell embryosNo probeanti-sense ssRNANo RNAdsRNA391:806, 1998 302006年的诺贝尔生理学奖获得者：Andrew Z. FireCraig C. Mello发现于19

14、9831Importance 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 th

15、e 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.322. Short RNAs that silence genes are produced from a variety of sources and direct the silencing of genes in three different w

16、ays 机制Short RNAs (functional input)33Different 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RNAsiRNAs & miRNAspre-miRN

17、As35Trigger 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).Three ways of the RNAi-directed gene silencing (functional output)36The heart of the RNAi mechanismDicer: an RNaseIII-like multidomain ribonucle

18、ase 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 express

19、ion.RNAi machinery 37Dicer:Structural organization:-A PAZ domain, binds the end of the dsRNA-Two RNase III domains-Other non-conserved domains.贾第鞭毛虫38RISC: the key component is Argonaute (AGO)Argonaute (AGO): A large protein family that constitutes key components of RISCs. -AGO proteins are characte

20、rized 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 acti

21、vity. -Distinct AGO members have distinct functions. For example, human AGO2 programs RISCs to cleave themRNA target, whereas AGO1 and AGO3 do not.39Topic 2: miRNA discoveryCHAPTER 18 RNAi and miRNA regulation一、miRNA发现的背景和miRNA发现40The discovery of miRNAs miRNA was first discovered in 1993 by Victor

22、Ambros at Harvard (lin-4) The second miRNA Let-7 was discovered in 2000 by Frank Slack as a postdoc at Harvard (Ruvkun lab)Victor AmbrosGary Ruvkun41The first discovered miRNA lin-4 in 1993Ruvkun G, Wightman B, Ha I. The 20 years it took to recognize the importance of tiny RNAs. Cell. 2004 Jan 23;11

23、6 (2 Suppl):S93-6.Lee R, Feinbaum R, Ambros V. A short history of a short RNA. Cell. 2004 Jan 23;116 (2 Suppl):S89-92Thought to be an oddity not a general phenomenon4243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。 44microRNAs had been neglected for so many year

24、s because of their small size. The underlying reason is: people never dream that small RNAs will have important biological roles. 45Topic 3: miRNA biogenesis and regulation三、miRNA生成和调控CHAPTER 18 RNAi and miRNA regulation461. MicroRNA (miRNA) & its processing微小RNA及其加工47MicroRNA (miRNA): A type of non

25、-coding small RNA (2123 nucleotides) produced by Dicer from a stem-loop structured RNA precursor (70-90 nts long) (结构和来源). miRNAs are widely expressed in animal and plant cells and functions in the form of RNAprotein complexes, termed miRISCs. miRNAs have been implicated in the control of developmen

26、t 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).48The miRNA genes and Structure of pri-miRNAsPri-miRNAs bear the 5 cap and 3 poly(A) tails49miRNA processingPri-miRNA(miRNA初级转录产物)Drosha(1)pre-miRNA(miRNA前体)Dicer(2) miRNAExporti

27、n 5 (Exp5) transports pre-miRNA to the cytoplasm50 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.51Han et al., Cell 125, 887901, June 2, 200652Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain.532. MicroRNA (miRNA) targeting and regulation.54A comparison