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1、microRNA的调控,RNA干扰造成的基因沉默,Gene knock-outs:remove the function of a gene permanently by disrupting its DNA;Gene silencing:suppress gene function by destroying or inhibiting mRNA(introducing RNA either binds to the mRNA and prevents it from being translated,or targets a nuclease to bind to the mRNA and
2、 degrade it)RNA interference(RNAi)recruits a natural RNA-degrading mechanism that is apparently ubiquitous in multicellular eukaryotes from plants to mammals.The phenomenon was first uncovered in plants during transgenic experiment:mRNA rapidly degraded.,2006年的诺贝尔生理学奖获得者：,Andrew Z.FireCraig C.Mello,
3、双链RNA抑制含其同源序列基因的表达,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,Why exogenous dsRNA can inhibit expression of gene
4、s homologous to that RNA?,从双链RNA产生的小干扰(small interfering)RNA(siRNAs)可以指导用不同机制关闭基因的细胞机器,RNAi silencing,Exogenous dsRNA外源双链RNA,RNA 干扰造成的基因沉默,Dicer:double-stranded RNAs to short interfering RNA(siRNA)siRNA incorporated into a nuclease-containing protein complex known as RISC(RNA-induced silencing compl
5、ex,including a ribonuclease called Argonaute);siRNA acts a“guide RNA”to target RISC to any mRNA containing an exactly complementary sequence.The mRNA is then degraded by the nuclease in the RISC.,The targets of the RNAi-directed gene silencingDegradation of the target mRNA(引起靶标mRNA的降解),Inhibition of
6、 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).,miRNA processing:Drosha,Dicer,Core of the RNAi mechanismDicer:an RNaseIII-like multidomain ribonuclease that first processes input dsRNA into small fragments called short interfering
7、 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.,Dicer:,Structural organization:-A PAZ dom
8、ain,binds the end of the dsRNA-Two RNase III domains-Other non-conserved domains.,RISC:the key component is Argonaute(AGO),AGO2,Dicer,TRBP,The multiple functions of RNAi,MicroRNA(miRNA),A type of non-coding small RNA(2123 nucleotides)Produced by Dicer from a stem-loop structured RNA precursor(70-90
9、nt)(结构和来源).miRNAs are widely expressed in animal and plant cells as RNAprotein complexes,termed miRISCs,and 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).,microRNA 介导的基因沉默,miRN
10、A were first identified in C.elegans as the products of the genes let-7 and lin-4,which control the timing of development.The role is similar with siRNA.1%of human genes code for miRNA.double-stranded RNA hairpin,the pre-miRNA,cleaved by Dicer produce a short single-stranded miRNA(22 nuleotides),RIS
11、C,miRNA acts a“guide RNA”to target RISC to any mRNA containing an exactly complementary sequence.,microRNA 介导的基因沉默,Unlike siRNA,animal miRNA are not perfectly complementary to their target mRNA and have a few mismatched bases.Once bound,the protein complex renders the mRNA inactive and suppresses tr
12、anslation,but not degraded.,Structure of pri-miRNAs,Pri-miRNAs bear the 5 cap and 3 poly(A)tails,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,Human Drosha and Dicer share the same RNase III domains and dsRNA bin
13、ding domain.,Victor R.Ambros,秀丽线虫 C.elegans,miRNA in C.elegans development,Expression of lin-4 allows C.elegans to proceed to the late developmental stage,miRNAs in vertebrate development:,Expression of miR-124a and miR-1 in Zebrafish,Medaka,Mouse,and Fly.miR-124a is restrictedly expressed in the br
14、ain 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,(Science 2004),3种miRNA控制造血干细胞向淋巴细胞的分化过程,miRNA controls the different
15、iation of the hematopoietic stem cell(调控造血干细胞的分化),Comparison between normal and tumor samples reveals global changes in miRNA expression.,Overexpression of the mir-1719b cluster accelerates c-myc-induced lymphomagenesis in mice.,siRNA application in mammalian,Transfect exogenous siRNA into cells(tra
16、nsient expression)Chemical synthesis:expensiveIn vitro transcription of pre-miRNA with T7 promoter.In vitro transcription of long dsRNA by that are then cleaved by E.coli RNase III or RNase III-like DICER.Expression of siRNA in cultured cells or in animal modelssiRNA produced with pol III promoter f
17、rom the transfected DNA plasmids.,Create induced phenotypes that can be observed over long time spans Create a stably engineered cells can be assayed either in vitro or in vivo,perhaps testing the angiogenic(血管生成)or metastatic(转移)potentials of tumor cells in xenograft models(异种移植模型)。Combine shRNAs w
18、ith existing high-efficiency gene delivery vehicles to create bona fide RNAi-based therapeutics.For example,ultimately,to silence a disease-causing mutant allele specifically.,Research Applications of RNAi:A new strategy of reverse genetics&a novel way of gene knock-out,It can be used in reverse gen
19、etics(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.,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.,
