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1、microRNA的调控的调控RNA干扰造成的基因沉默干扰造成的基因沉默l 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 bin
2、d to the mRNA and degrade it)l RNA interference (RNAi) recruits a natural RNA-degrading mechanism that is apparently ubiquitous in multicellular eukaryotes from plants to mammals.l 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：年的诺贝
3、尔生理学奖获得者：双链双链RNA抑制抑制基因的表达基因的表达 (in situ hybridization in embryos) (胚胎的原位杂交胚胎的原位杂交).No hybridization and staining+ hybridization (endogenous mex-3 RNA)+antisense+hybridization+ds mex-3 RNA+hybridization从双链从双链RNA产生的小干扰产生的小干扰 (small interfering) RNA (siRNAs) 可以指导用可以指导用不同机制关闭基因的细胞机器不同机制关闭基因的细胞机器RNAi sil
4、encingExogenous dsRNA外源双链外源双链RNARNA 干扰造成的基因沉默干扰造成的基因沉默l Dicer: double-stranded RNAs to short interfering RNA (siRNA)l siRNA incorporated into a nuclease-containing protein complex known as RISC (RNA-induced silencing complex, including a ribonuclease called Argonaute); siRNA acts a “guide RNA” to ta
5、rget 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 silencing1.Degradation of the target mRNA (引起靶引起靶标标mRNA的降解的降解),2.Inhibition of translation of the target mRNA (抑制靶标抑制靶标mRNA的翻译的翻译), 3.Silenc
6、ing the gene transcription from the target promoter (引起靶标启动子的转录引起靶标启动子的转录沉默沉默).l miRNA processing: Drosha, Dicer Core of the RNAi mechanism an RNaseIII-like multidomain ribonuclease that first processes input dsRNA into small fragments called short interfering RNAs (siRNAs) or microRNAs (miRNA). Dic
7、er then helps load its small RNA products into RISC.: a large multiprotein complex that direct the bound siRNA or miRNA to its target and inhibit the target gene expression.Structural organization:-A PAZ domain, binds the end of the dsRNA-Two RNase III domains-Other non-conserved domains.The multipl
8、e functions of RNAiMicroRNA (miRNA)l A type of non-coding small RNA (2123 nucleotides) l Produced by Dicer from a stem-loop structured RNA precursor (70-90 nt) (结构和来结构和来源源). l miRNAs are widely expressed in animal and plant cells as RNAprotein complexes, termed miRISCs, and have been implicated in t
9、he control of development because they lead to the destruction or translational suppression of target mRNAs with homology to the miRNA (生生物学功能和机制物学功能和机制).microRNA 介导的基因沉默介导的基因沉默l miRNA were first identified in C. elegans as the products of the genes let-7 and lin-4, which control the timing of devel
10、opment. The role is similar with siRNA. 1% of human genes code for miRNA.l double-stranded RNA hairpin, the pre-miRNA, cleaved by Dicer produce a short single-stranded miRNA ( 22 nuleotides), RISC, miRNA acts a “guide RNA” to target RISC to any mRNA containing an exactly complementary sequence. micr
11、oRNA 介导的基因沉默介导的基因沉默l 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 translation, but not degraded. Pri-miRNAs bear the 5 cap and 3 poly(A) tailsPri-miRNA(miRNA初级
12、转初级转录产物录产物)pre-miRNA(miRNA前体前体) miRNAExportin 5 (Exp5) transports pre-miRNA to the cytoplasmHuman Drosha and Dicer share the same RNase III domains and dsRNA binding domain.Victor R. Ambros秀丽线虫秀丽线虫 C. elegansmiRNA in C. elegans development allows C. elegans to proceed to the late developmental stage
13、 miRNAs in vertebrate development: 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. 青鳉青鳉斑马鱼斑马鱼小鼠小鼠果蝇果蝇Learning the miRNA function from its ex
14、pression pattern( Science 2004)3种种miRNA控制造血干细胞向淋巴细胞的分化过程控制造血干细胞向淋巴细胞的分化过程miRNA controls the differentiation of the hematopoietic stem cell (调控造血干细胞调控造血干细胞的分化的分化)Comparison between normal and tumor samples reveals global changes in miRNA expression. siRNA application in mammalian (transient expressio
15、n)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.siRNA produced with pol III promoter from the transfected DNA plasmids.1. Create induced phenotypes that can
16、be observed over long time spans 2. Create a stably engineered cells can be assayed either in vitro or in vivo, perhaps testing the angiogenic (血管生成血管生成) or metastatic (转移转移) potentials of tumor cells in xenograft models (异种移植模型异种移植模型)。 3. 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. Research Applications of RNAi: A new strategy of reverse genetics
