【正文】 RNAi 毛自朝 農(nóng)學(xué)院生物技術(shù)系 Tel: 5227732 13114297551 Outline of this talking ? Introducing of gene silence ? History and definition of RNA ? Molecular mechanism of RNAi ? Main ponents of RNAi ? Biological Function of RNAi ? Mechanism of DNA methylation by RNAi inducing ? Example Translation regulation by RNAi ? Unanswered question ? Application Regulate Gene expression level ? DNA (pretranscription ) gene amplification ? Transcriptional level (promoter, TF. Enhancer Reducer ) ? Post Transcriptional Level (mRNA ,tRNA and rRNA maturation Spicing Exton, Cap and polyA, RNAi) ? Translational and post translational level Other names of posttranscriptional gene silencing (PTGS) : – gene silencing – RNA silencing – RNA interference – In certain fungi: quelling RNAi can spread throughout certain anisms (C. elegans, plants). Why is RNAi important? Most widely held view is that RNAi evolved to protect the genome from viruses (or other invading DNAs or RNAs) Recently, very small (micro) RNAs have been discovered in several eukaryotes that regulate developmentally other large RNAs –May be a new use for the RNAi mechanism besides defense Recent applications of RNAi Modulation of HIV1 replication by RNA interference. Hannon(2022). Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. An et al.(1999) Selective silencing of viral gene expression in HPVpositive human cervical （ 頸 ） carcinoma cells treated with siRNA, a primer of RNA interference. Jung et al. 2022. RNA interference in adult mice. Mccaffrey et Successful inactivation of endogenous Oct3/4 and cmos genes in mouse pre implantation embryos and oocytes using short interfering RNAs. Le Bon et Posttranscriptional gene silencing Promoters active Gene hypermethylated in coding region Purpose Viral immunity? S. Grant (1999) Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS) This has recently been termed ―RNAi‖ Promoters silenced Genes hypermethylated in promoter region Purpose Viral immunity? Short history of RNAi Definition: the ability of exogenous doublestranded RNA (dsRNA) to suppress the expression of the gene which corresponds to the dsRNA sequence. 1990 : Introduction of transgenes homologous to endogenous genes often resulted in plants with both genes suppressed! Called Cosuppression Resulted in degradation of the endogenous and the transgene mRNA (Plant） ? 在 1990年， Napoli等試圖用轉(zhuǎn)基因技術(shù)使有色矮牽?；?(petunias)的花朵更加艷麗，他們將色素合成相關(guān)基因 (CHS基因 )置于一個(gè)強(qiáng)啟動(dòng)子(CaMV35S)之下，轉(zhuǎn)人矮牽牛花，希望通過在該植物花中增加色素基因的拷貝數(shù)，使該植物開出更艷麗的花朵。然而，實(shí)驗(yàn)結(jié)果則與預(yù)期相反：不少花朵的顏色減退，有的甚至變成了白色花朵。接下來的深入研究證明，被轉(zhuǎn)入該植物花中的外源色素基因的表達(dá)被抑制，轉(zhuǎn)基因植物本身的同源色素基因的表達(dá)也受到抑制，這種外源基因和本身的同源基因均受到抑制的現(xiàn)象被稱做共抑制(CO—suppression)，但當(dāng)時(shí)該現(xiàn)象的本質(zhì)不明? 。 ? 1994年，當(dāng) Cogoni和其同事以真菌脈孢霉(Neurospora)為實(shí)驗(yàn)材料把外源基因轉(zhuǎn)人該菌時(shí)，該菌本身的基因表達(dá)也受到抑制，當(dāng)時(shí)把這種在真菌中發(fā)現(xiàn)的類似現(xiàn)象稱為基因消除 (quelling)，但當(dāng)時(shí)仍未明確其機(jī)制。 Fungi 線蟲 C. elegans ? 1995年，美國康乃爾大學(xué)的 Su Guo在以線蟲 (Caenorhabditis elegans)為實(shí)驗(yàn)材料，試圖用反義 RNA技術(shù)阻斷線蟲 par—l基因的表達(dá)以探索該基因的功能時(shí)發(fā)現(xiàn)：當(dāng)向線蟲的胚細(xì)胞中注入與該基因 mRMA互補(bǔ)的反義 RNA時(shí)，該基因的表達(dá)如預(yù)期的那樣被阻斷。但是在注人與該基因 mRMA相同的正義 RNA的對照組中，該基因的表達(dá)也同樣被阻斷，這一現(xiàn)象無法用傳統(tǒng)的反義RNA技術(shù)的機(jī)制來解釋。 1995 Guo and Kemphues: injection of either antisense or sense RNAs in the germline of C. elegans was equally effective at silencing homologous target genes 1998 Mello and Fire: extension of above experiments, bination of sense and antisense RNA (= dsRNA) was 10 times more effective than single strand RNA uninjected, mex3 probe uninjected, no probe antisense mex3 RNA, mex3 probe doublestranded mex3 RNA injected, mex3 probe Doublestranded RNAinduced RNA interference causes destruction of a specific mRNA in C. elegans Guo, S. and Kemphues, K. J. Cell 81, 611620 (1995) Fire, A. et al. Nature 391, 809 (1998) Key points of C. elegans experiment ?Substoichiometric amounts of dsRNA relative to the targeted mRNA are required to pletely eliminate the mRNA (. the dsRNA is catalytic) ?dsRNA is 10100X better than antisense or sense RNA ?Doesn’t work if introns or promoters are targeted by the dsRNA ?Doesn’t interfere with transcription initiation or elongation (it is possible to target a single gene in an operon) (. RNAi is a posttranscriptional phenomena) ?The targeted mRNA is degraded (. it can’t be detected by probes) ?dsRNA can cross cellular boundaries (. there is a transport mechanism) RNAi works in other anisms Zamore, P. D. Science 296, 12651269 (202