分子生物学英文课件：lecture10+regulatory+RNAs

大连理工大学Molecular BiologyChapter 18 Regulatory RNAs大连理工大学Molecular BiologyPart IV Regulation Ch 16: Transcriptional regulation in prokaryotes Ch 17: Transcriptional regulation in eukaryotes Ch18: Regulatory RNAs TOPIC 1 Regulation by RNAs in Bacteria TOPIC 2 Regulatory RNAs are Widespread in Eukaryotes TOPIC 3 Synthesis and Function of miRNA Molecules TOPIC 4 Silencing Gene Expression by Small RNAs TOPIC 5 Long Non-Coding RNAs Ch 19: Gene regulation in development and evolution Ch 20: Genome Analysis and Systems BiologyCh18RNA调控调控miRNAlncRNA真核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyTOPIC 1 Regulation by RNAs in BacteriaSmall RNAs have been recognized in prokaryotes for many years. Some are involved in regulating the replication of plasmids, and others are involved in regulating gene expression Control transcriptionthe 6S RNA of Escherichia coli. This RNA binds to the s70 subunit of RNA polymerase and down-regulates transcription from many s70 promoters.In recent years, attention has focused on small RNA molecules in bacteria that regulate translation and mRNA degradationSmall RNAs (sRNA): regulation by base pairing. A well-studied sRNA from E. coli is the 81-nucleotide RybB RNA. This sRNA binds several target mRNAs and triggers their destruction by the nuclease RNase E.“antisense” RNAsRiboswitches (核糖开关核糖开关): regulation by metabolite-mediated structure changes, alternative RNA pairing that truly operate in cisAttenuation（衰减作用）（衰减作用）: Regulation by ribosome stop-mediated formation of terminators Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular Biology1.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 Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular Biology2.RiboswitchesRiboswitches are regulatory RNA elements that act as direct sensors of small molecule metabolites to control gene transcription or translation.Perhaps the simplest bacterial RNA regulatory elements . These regulatory elements are typically found within the 5-untranslated regions (5-UTRs) of the genes, and they can regulate expression at the level of transcription or translation by adopting different conformations in response to environmental signals, including stalled ribosomes, uncharged tRNAs, elevated temperatures, or small molecule ligands. TargetsReside upstream of the targeted mRNA, and form specific structure to bind its small molecule ligand. MechanismAct in cis by alteration of its own structure upon the binding of the small metabolites. MechanismCh18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThe structure of a riboswitch in its regulated mRNAEach riboswitch is made up of two components: the aptamer (适配适配体体) and the expression platform（表达平台）（表达平台）.The aptamer binds the small-molecule ligand and, in response, undergoes a conformational change, which, in turn, causes a change in the secondary structure of the adjoining expression platform. These conformational changes alter expression of the associated gene by either terminating transcription or inhibiting the initiation of translation.Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyRiboswitches regulate Control of transcription termination by a riboswitch(left) Control of translation initiation by a riboswitch（right）代谢物代谢物代谢物代谢物 Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyAlteration of of the structure of the SAM riboswitchCh18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular Biology焦磷酸硫胺素大连理工大学Molecular Biology3. Attenuation (衰减作用衰减作用) 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. mechanismCh18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular Biology大连理工大学Molecular BiologyImportance of attenuationA typical negative feed-back regulationUse of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan.Attenuation alone can provide robust regulation.Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead.Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyRNAs as Defense Agents in Prokaryotes and ArchaeaAlthough it is not strictly an example of gene regulation (it is a system of defense against viruses and other extrachromosomal intruders入侵), the mechanism used is strikingly similar to systems we will encounter in eukaryotes, namely, RNAi.CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats，成簇的、 有规律的、间隔的、短回文重复序列) are a record of infections survived and resistance gained repeat：each 30 bp long and highly conserved within a given cluster spacer：similar length of repeat but highly divergent sequence leader sequence：often A-T rich and 500 bp in lengthCh18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyHow do CRISPRs arise, andwhat do they do? These clusters are not rarein half of all bacterial genomes sequenced, and essentially all genomes of Archaea. In many cases, there is only one cluster per genome, but can range up to 20 or more,eg. 400 in Chloroflexus(绿曲挠丝状菌). CRISPR functions as a prokaryotic immune system( acquired immunity). , confers resistance to exogenous genetic elements such as plasmids and phages. Short segments of foreign DNA, spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures.CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyCRISPR functions as a prokaryotic immune system Spacer sequences are acquired from infecting viruses CRISPR is transcribed as a single long RNA, which is then processed into shorter RNA species that target destruction of invading DNA or RNAPAM (proto-spacer adjacent motif) : the sequence in the virus that will become a new spacer. When a new spacer is added to a CRISPR array, it is incorporated at the proximal end(近端), near the leader sequence.Ch18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular Biologystructure of the CASCADE complex in E. coli.In the case of E. coli, the CRISPR is associated with eight cas genes, the products of five of which form a complex called Cascade.This complex includes one subunit that is implicated in the processing of the long transcript into the individual short crRNAs, each the length of one spacer and one repeat sequence.These small RNAs remain bound to the Cascade complex and direct it to the DNA genomes of invading foreign DNACh18RNA调控调控miRNAlncRNA真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyIn general, operation of the CRISPR/cas system takes place in four steps1. Spacers corresponding to fragments of DNA from a phage or another source are incorporated into CRISPR. It is believed to involve the Cas1 and Cas2 proteins. Also, new spacers are added at the leading end of the CRISPR sequence.2. CRISPR loci must be transcribed into pre-crRNA. A noncoding sequence at the leading end of CRISPR, rich in adenine and thymine, acts as a promoter for this purpose.3. Pre-crRNA must then be processed into crRNA. Each consists of a single spacer between two half-repeats.4. After processing, the crRNA is used to neutralize foreign genetic material.The CRISPR/cas system is similar in principle to the RNA interference used by eukaryotic cells. However, they employ entirely different sets of proteins. No homology between CRISPR/cas and RNAi .大连理工大学Molecular BiologyTOPIC 2 RNA Interference Is a Major Regulatory Mechanism in Eukaryotes Regulatory RNAs are widespread in eukaryotes.PiRNA: are expressed predominantly in the germline and have features distinct from miRNAsCh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyRNA interference and its mechanism1. Double-stranded RNA inhibits expression of genes homologous to that RNA. 双链RNA抑制含其同源序列基因的表达phenomena-现象现象2. Short interfering RNA (siRNAs) are produced from dsRNA and direct machinery that switch off genes in various way.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Mechanism-机制机制Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyCover page story: Chapter 18 Regulatory RNAsAround 1990, molecular biologists obtained a number of unexpected results that were difficult to explain. The most striking effects were observed by plant biologists who were trying to increase the colour intensity of the petals in petunias(矮牵牛) by introducing a gene inducing the formation of red pigment in the flowers. But instead of intensifying the colour, this treatment led to a complete loss of colour and the petals turned white! 紫丁香的故事: 2006诺贝尔医学奖缘于爱情Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyTwo other contributorsProf. Su GuoNobel Laureate Andrew Fire highlighted experiments Guo launched when she was a Cornell graduate student. http:/genetics.wustl.edu/tslab/http:/bts.ucsf.edu/guo/home.htmlCh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyAnalysis of RNA-interference effects in individual cells大连理工大学Molecular BiologyEffects of mex-3 RNA interference on levels of the endogenous mRNA: in situ hybridization of 4-cell stage embryos(A) Negative control showing lack of staining in the absence of the hybridization probe. (B) Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA (purple staining). (C) Embryo from parent injected with purified mex-3 antisense RNA. These embryos (and the parent animals) retain mex-3 mRNA, although levels may be somewhat less than wild type. (D) Late 4-cell stage embryo from a parent injected with dsRNA corresponding to mex-3 ; no mex-3 RNA is detected. (Templates used for interfering RNA and in situ probes were largely non-overlapping.) Each embryo is approximately 50 m in length.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThe discovery of RNAi explains the virus-induced gene silencing in plants (植物病毒引起的基因沉默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.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyDouble-stranded RNA can be introduced experimentally to silence target genes of interestIn plants, silencing can be triggered, for example, by engineered RNA viruses or by inverted repeat transgenes. In worms, silencing can be triggered by injection or feeding of dsRNA. In both of these systems, silencing is systemic and spreads throughout the organism. a, A silencing signal moves from the veins into leaf tissue. Green is green fluorescent protein (GFP) fluorescence and red is chlorophyll fluorescence that is seen upon silencing of the GFP transgene. b, C. elegans engineered to express GFP in nuclei. Animals on the right have been treated with a control dsRNA, whereas those on the left have been exposed to GFP dsRNA. Some neuronal nuclei remain florescent, correlating with low expression of a protein required for systemic RNAi59. c, HeLa cells treated with an ORC6 siRNA and stained for tubulin (green) and DNA (red). Depletion of ORC6 results in accumulation of multinucleated cells. Stable silencing can also be induced by expression of dsRNA as hairpins or snap-back RNAs. d, Adult Drosophila express a hairpin homologous to the white gene (left), which results in unpigmented eyes compared with wild type (right).Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyWhy exogenous dsRNA can inhibit expression of genes homologous to that RNA?Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThree ways of the RNAi-directed gene silencing1. 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).Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyRNAi mechanism 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. (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.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyDicer： the molecular ruler 贾第鞭毛虫Structural organization:A PAZ domain, binds the end of the dsRNATwo RNase III domainsOther non-conserved domains.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThe crystal structure of Dicer, a module that binds the end of dsRNA, is separated from the 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.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyRISC: the key component is Argonaute (AGO): A large protein family that constitutes key components of RISCs. AGO proteins are characterized by 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. . For example, human AGO2 programs RISCs to cleave the mRNA target, whereas AGO1 and AGO3 do not.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThe crystal structure of Argonaute Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyA model for siRNA-guidedmRNA cleavage by ArgonauteCh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyRNAi silencing is extremely efficient Very small amounts of dsRNA are often enough to induce a near complete shutdown of target gene expression. A factor adding to efficiency could be the action of an RNA-dependent RNA Polymerse (RdRP ), an additional enzyme required in many cases of RNAi. This polymerase can amplify the inhibitory signal: the RdRP generates dsRNA after recruitment to the mRNA by the original siRNA. This feedback process generates large amounts of siRNA. RdRP has not yet been identified in mammalian cells, high efficiency there likely results from the fact that slicing is catalyticeach RISC can cleave several mRNAs.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyAmplification of thesiRNA signal by RdRP. 1. As shown in Figure, the siRNA signal can be amplified, generating more dsRNA for Dicer to process into more siRNAs.2. This is achieved because the siRNARISC complex can recruit an enzyme, RNA-dependent RNA polymerase to the targeted RNA, and the siRNA acts as a primer for that enzyme to transform the target into dsRNA, which can itself then be acted on by Dicer. 3. RdRPs are found in plants, worms, and the yeast Schizosaccharomyces pombe (Saccharomyces cerevisiae does not have the RNAi machinery at all).Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyIn plants, RdRPs (including RDR6) use the cleaved transcript fragments as templates to synthesize long dsRNA; the dsRNA is then diced into secondary siRNAs. Secondary siRNAs are formed by DICER-LIKE 4 (DCL4) both 5 and 3, suggesting that mRNA cleavage, rather than priming of RdRP by primary siRNAs, is the signal for siRNA amplification. Secondary siRNAs in Arabidopsis might sometimes be primed. Nature Reviews Genetics 10, 94-108 (February 2009) | doi:10.1038/nrg2504 Caenorhabditis elegans uses a different mechanism. In worms, primary siRNAs are bound to RDE-1, a primary Argonaute. The primary siRNAs guide RDE-1 to the target mRNA, to which it recruits RdRPs that synthesize secondary siRNAs. Worm secondary siRNAs have a 5 diphosphate or triphosphate, indicating that they are produced by transcription rather than by dicing and, at least in vitro, secondary siRNA production does not require Dicer. RdRP（eg. QDE1） can directly transcribe short RNA oligomers of 22 nucleotides from a much longer template.大连理工大学Molecular BiologyThe multiple functions of RNAiCh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyTOPIC 3 Synthesis and Function of miRNA MoleculesmiRNA 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 (Gary Ruvkun lab)Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThe number of the identified miRNAs is growing rapidly in recent yearshttp:/www.mirbase.org/大连理工大学Molecular BiologymiRNAs have a characteristic structure that assists in identifying them and their target genesThe functional form of an miRNA is typically 21 or 22 nucleotides (it can vary from 19 to 25 nucleotides). miRNA s are generated by cleavage from a longer RNA transcript.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologymiRNA biogenesis and regulation MicroRNA (miRNA): A type of non-coding small RNA (2123 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 RNAprotein 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Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologymiRNAs are coded inboth introns and exons in RNACh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyThe miRNA are generated by two RNA cleavage reactionsThese short RNAs are generated by two RNA cleavage reactionsThe first cleavage liberates the stem-loop, called the pre-miRNA; The second generates the mature miRNA from the pre-miRNACh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologymiRNA processingCh18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyHuman Drosha and Dicer share the same RNase III domains and dsRNA binding domainThe RNase III family enzymes are specific for dsRNA and cleave it in a manner that leaves a 2-nucleotide overhang on the 3 ends of the dsRNA product. This 3 overhang is important for recognition of that RNA molecule by the next enzyme in the pathway, Dicer. In plants, miRNAs are generated by Dicer alone; it is not clear how they manage to forgo prior action of Drosha.Ch18RNA调控调控miRNAlncRNA真核机制真核机制原核机制原核机制siRNA小 结练 习大连理工大学Molecular BiologyDrosha-catalyzed cleavageThe pre-miRNA generated by Drosha is usually 6570 nucleotides. Drosha resides in nucleus, and there Drosha-catalyzed cleavage event occurs.The base-paired stem in the pri-miRNA is typically 33 bp in length(three helical turns of dsRNA), with a terminal loop and flanking segments and contains only a few mismatches.The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. The cleavage site is dete