【正文】 2) RNAi in other anism ? 研究人員又在果蠅 (Drosophila)、渦蟲 (Planaria)、椎蟲 (Trypanosomes)、無脊椎動物、脊椎動物、哺乳動物 (由短于 30 bp的 dsRNA誘發(fā) RNAi )、植物等許多真核生物中發(fā)現(xiàn)了 RNAi作用。經(jīng)幾年研究，研究者們已認(rèn)識到： RNAi是在動、植物分化以前就已普遍存在的一種古老的生物學(xué)現(xiàn)象，是真核生物體抵抗外源基因 (如病毒基因、轉(zhuǎn)座子、人工轉(zhuǎn)入基因等 )入侵的一種保護性反應(yīng)，因此又被稱為基因組的免疫機制。同時，它還是生物體在不同時期通過調(diào)控基因表達來調(diào)節(jié)細(xì)胞分化的機制。 silencing of GFP in leaf veins silencing of GFP in C. elegans nuclei depletion of ORC6 results in multinucleated HeLa cells depletion of White results in unpigmented Drosophila eyes RNAi works in other anisms Hannon, G. J. Nature 418, 244251 (2022) SIN3 p55 218 131 86 Synthesize ~800 bp dsRNA in vitro Add ~50?g of dsRNA to 2X106 cells Incubate at room temperature ~3 days Harvest cells for protein, RNA, Elimination of SIN3（ histone deacetylases ） by RNAi in Drosophila tissue culture cells Western blot (loading control) (RNAi target) SIN3 RNAiSIN3 ControlSIN3 ControlDAPI SIN3 RNAiDAPI RNAi works in an all or none fashion Immunofluorescence of Drosophila S2 cells with or without SIN3 RNAi Wt CUC1/2 syn miRNA Synthetic miRNAs 定義 ? RNA干涉 (RNA interference， RNAi)是指雙鏈RNA(double—strand RNA， dsRNA)在細(xì)胞內(nèi)特異性地誘導(dǎo)與之同源互補的 mRNA的降解，使相應(yīng)基因的表達關(guān)閉，從而引發(fā)基因轉(zhuǎn)錄后水平沉默的現(xiàn)象 (Post Transcriptional gene silencing，PTGS) 。 ? 與 RNAi有關(guān)的 dsRNA及蛋白質(zhì)可參與染色質(zhì)的修飾作用，使其中的組蛋白和 DNA發(fā)生甲基化作用，使相應(yīng)基因不能被轉(zhuǎn)錄，從而導(dǎo)致受阻基因不能表達。這種在轉(zhuǎn)錄水平上阻斷基因功能，使基因沉默的 RNAi方式被稱為轉(zhuǎn)錄基因沉默(Transcriptional gene silencing， TGS)。 Definition dsRNA 5’ 5’ RNAinduced silencing plex (active, 100kDa plex) small interfering RNAs Mechanism of RNAi posttranscriptional gene silencing (inactive, 250500kDa plex) (endonucleolytic cleavage in the region of homology) (a critical step in the activation of RISC) Zamore, P. D. Science 296, 12651269 (2022) Dicer contains 5 domains ? 2 catalytic RNase domains ? a dsRNAbinding domain ? a helicase domain ? a PAZ domain Dicer is thought to work as a dimer One of the catalytic sites in Dicer is defective Thus, instead of cleaving from ~911 nt, like bacterial RNase III Dicer cleaves ~22 nt (see panel B) Hannon, G. J. Nature 418, 244251 (2022) Dicer RISC contains at least 4 subunits ?Argonaute (5 homologs in Dros.) ?dFXR (the Dros. homologue of human fragile X mental retardation protein) ?Vasa intronic gene (VIG) ?nuclease Activated RISC uses the unwound siRNA as a guide to substrate selection RISC Hannon, G. J. Nature 418, 244251 (2022) ? 在 Argonaute蛋白的協(xié)助下， Dicer酶識別外源的長的雙鏈 RNA分子 (dsRNA)，并以一定的間隔將dsRNA切成 22個核苷酸大小的小的干涉 RNA分子(siRNA)． siRNA隨即與效應(yīng)物 RISC(RNA誘導(dǎo)的沉默復(fù)合物 )結(jié)合，在 ATP的作用下解旋，從而使RISC由 250 kD大小的前體形式變成約 100 kD左右的 RISC活性形式 (RISC*)．通過堿基互補配對的原則， siRNA識別與自身具有同源序列的內(nèi)源靶 mRNA，最后通過 RISC*將 mRNA降解，從而達到阻止靶基因表達、使基因沉默的目的． 二、 RNAi的分子機制 ? RNAi發(fā)生的基本過程可分為起始階段和效應(yīng)階段 。 ? 在起始階段， RNaselII家族的雙鏈特異的核糖核酸酶 (在果蠅中命名為 Dicer)以 ATP依賴的方式將長的 dsRNA切割成 21—23 nt的小干擾 RNA(small interfering RNA， siRNA) 。 ? 在效應(yīng)階段， siRNA參與形成 RNA誘導(dǎo)的沉默復(fù)合物 (RNA—induced silencing plex， RISC) 。 二、 RNAi的分子機制 ? RISC以 ATP依賴的方式催化雙鏈 siRNA解旋 。然后利用其內(nèi)部的單鏈 siRNA／miRNA，通過堿基配對識別與之互補的靶RNA 。 RISC中的核酸內(nèi)切酶在距離 siRNA 3’端 12個堿基的位置切割靶 RNA 。 ? 切割后的靶 RNA在核酸外切酶的作用下被降解掉，導(dǎo)致目的基因的沉默． ? a | The RNA induced silencing plex (RISC) catalyses a phosphodiester hydrolysis reaction that generates 3′hydroxyl and 5′phosphate termini. The nucleotides of the target mRNA that are paired to the short interfering RNA (siRNA) nucleotides 10 and 11 (counting from the siRNA 5′ end) ? b | Target cleavage by RISC is not affected by a 2′deoxyribose (H) substitution at the cleavage site (scissile bond), but 2′Omethyl ribose (OCH3) and phosphorothioate (S) substitutions at the same phosphodiester linkage inhibit cleavage. ? C RISC activity requires divalent metal ions, and a sulphursubstitution experiment indicates that one or more metal ions (Me2+) might directly coordinate one or both of the nonbridging oxygen atoms of the phosphate。 Chemical mechanism of RISCcatalysed targetmRNA cleavage 裂解的化學(xué)機制 ?How does RNAi spread throughout and anism, even when triggered by minute quantities of dsRNA? Requires a system to pass a signal from one cell to another Requires a strategy for amplifying the signal Unanswered question A proposed model for amplification and spreading of silencing RNAdirected RNA polymerase RNAdirected RNA polymerase Zamore, P. D. Science 296, 12651269 (2022) Does not appear to occur in mammals, therefore the effects of siRNA are transient Transitive RNAi: silencing can travel 3’ to 5’ on a specific mRNA target Hannon, G. J. Nature 418, 244251 (2022) RNAi can silence gene expression thr