【正文】 ) Drosha (1) premiRNA (miRNA前體 ) Dicer (2) miRNA Exportin 5 (Exp5) transports premiRNA to the cytoplasm 132 1. A typical metazoan primiRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. 2. The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. 3. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stemssRNA junction. 133 Han et al., Cell 125, 887–901, June 2, 2022 134 Human Drosha and Dicer share the same RNase III domains and dsRNA binding domain. 135 2. MicroRNA (miRNA) targets and regulation. 136 A parison between miRNA and siRNA 137 Three strategies of miRNA and target recognition (targets are locating in 3’ UTRs). 138 Topic 3: miRNA roles in development, cell differentiation and virus MicroRNA在發(fā)育中的調(diào)控作用，及其他作用 139 Victor R. Ambros 秀麗線蟲 C. elegans 1. miRNA in C. elegans development 140 lin4 and let7 miRNAs control the developmental time of C. elegans. 141 Expression of lin4 allows C. elegans to proceed to the late developmental stage 142 lin4 binds its target mRNAs by imperfect base pairing. 143 2. miRNAs in vertebrate development: There are a lot unknown because the the lack of efficient methods to uncover the specific targets of miRNAs. 144 Figure 2. Expression of miR124a and miR1 in Zebrafish, Medaka, Mouse, and Fly. miR124a 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 miR1 is restricted to the muscles and the heart in the mouse. 青鳉 斑馬魚 小鼠 果蠅 Learning the miRNA function from its expression pattern 145 3. miRNA controls plant phenotypes and more (控制植物表型特征 ) JawmiRNA 控制擬南芥葉形變化 (Nature, 2022) 146 ( Science 2022) 3種 miRNA控制造血干細(xì)胞向淋巴細(xì)胞的分化過(guò)程 4. miRNA controls the differentiation of the hematopoietic stem cell (調(diào)控造血干細(xì)胞的分化 ) 147 5. Some viruses encode miRNAs (有些病毒編碼 miRNAs) 148 Topic 4: miRNA in cancer 微小 RNA在癌癥發(fā)生中的作用 149 miRNAs in human: There are over 700 miRNAs from human have been found and annotated so far. They are named as hasmiRx. 150 miRNA expression pattern changes during oncogenesis, and is unique for each cancer. 微小 RNA在癌癥發(fā)生中表達(dá)譜的變化 151 152 Figure 3, Comparison between normal and tumor samples reveals global changes in miRNA expression. 153 微小 RNA調(diào)控癌基因表達(dá)：抑制 or 激活？ ? 微小 RNA激活癌基因 154 1. cMyc is a helix–loop–helix leucine zipper transcription factor that regulates an estimated 10–15% of genes in the human and Drosophila genomes. 2. cMyc activates expression of a cluster of six miRNAs (mir1792 cluster) on human chromosome 13. (Figure 1) 3. E2F1 is the transcription factor, which is a target of cMyc that promotes cell cycle progression. 4. Expression of E2F1 is negatively regulated by two miRNAs in this cluster, miR175p and miR20a. (Figure 4) 155 156 Some microRNAs are potential oncogenes 有些微小 RNA可能是致癌基因。 157 B細(xì)胞淋巴瘤 158 Figure 1. The mir17–92 cluster shows increased expression in Bcell lymphoma samples and cell lines. The level of mir17–92 primiRNA was determined by realtime quantitative RTPCR in 46 lymphomas and 47 colorectal carcinomas, and pared to levels found in corresponding normal tissues from five individuals. 159 Figure 2. Overexpression of the mir17–19b cluster accelerates cmycinduced lymphomagenesis in mice. Dicer contains two RNAse III domains siRNAs long dsRNA 是細(xì)胞內(nèi)一類雙鏈 RNA（ dsRNA）在特定情況下通過(guò)一定酶切機(jī)制，轉(zhuǎn)變?yōu)榫哂刑囟ㄩL(zhǎng)度和序列的小片段 RNA 小干擾 RNA (small interfering RNA, siRNA) siRNA具有特定的結(jié)構(gòu) 19 nt duplex 2 nt 3’ overhangs 1990年， Jensen 等 RNA干擾 的發(fā)現(xiàn) 苯基苯乙烯酮合酶 (chalcone synthase) 共抑制（ cosuppression）現(xiàn)象 C. elegans silencing Overexpression 1995年， Guo 等 + + _ par1 mRNA antisense strand RNA sense strand RNA C. elegans par1 mRNA antisense strand RNA sense strand RNA silencing + + _ 1995年， Guo 等 ssRNA contaminant ssRNA silencing 1998年， Fire和 Mello － RNA ＋ RNA No silencing RNA interference（ RNAi） dsRNA silencing 1999年 Tuschl 等：在哺乳動(dòng)物中也存在 RNAi 2022年 Berstein 等 : 發(fā)現(xiàn)只有 22核苷酸的 siRNA（ small interfering RNA），同時(shí)他們還發(fā)現(xiàn)了體內(nèi)一個(gè)分解 dsRNA為 siRNA的叫 Dicer的酶 Andrew Z. Fire Craig C. Mello 什么是 RNA干擾 ? 由 siRNA介導(dǎo)的基因表達(dá)抑制作用稱為 RNA干擾。 ?靶基因同源的 dsRNA進(jìn)入胞內(nèi) ?任何與 dsRNA序列同源的 mRNA 將被沉默 nucleus dsRNA RAN干擾如何實(shí)現(xiàn) ? RNAi干擾是在轉(zhuǎn)錄后特異性降解 mRNA…… 關(guān)鍵兩步驟 ! dsRNA 被水解為 2123 nt 的小分子干擾 RNA 34 27 21 20 16 shortinterfering RNA QuickTime?and aGIF depressorare needed to see this picture.第一步 : Tuschl, 2022 Dicer屬于 RNase家族 ,專門將雙鏈 RNA切割成等長(zhǎng)的小片段 Tuschl, 2022 siRNA與蛋白質(zhì)形成 RISC，與特異 mRNA結(jié)合使其降解 第二步 : RNAinduced silencing plex, RISC siRNA RNAi干擾模式 30bp 雙鏈 RNA（ dsRNA） 水解生成 2123nt siRNA 5’ 3’ 3’ 5’ RISC形成 識(shí)別特異序列 并使其降解 RNA干擾作用機(jī)制 ? RNAi干擾過(guò)程錄像 siRNA application siRNA的應(yīng)用 siRNA application in mammalian Transfect exogenous siRNA into cells ? Chemical synthesis: expensive ? Generate esiRNA (enzymatic produced siRNA) from in vitro transcribed long dsRNA by E. coli RNase III or RNase IIIlike DICER. Expression of siRNA in cultured cells or in animal models ? siRNA produced with pol III promoter from the transfected DNA plasmids. 1. Transcription from RNAP III promoters of U6 and H1 are well characterized. RNAP III transcription uses a welldefined termination signal (TTTTT) and the products have no extra sequence. 2. Transcription from these promoters is very efficient in various tissues. Expression of hairpin RNA (shRNA) using a Pol III promoter. 通過(guò)將編碼 shRNA的質(zhì)粒轉(zhuǎn)染進(jìn)細(xì)胞內(nèi)。 轉(zhuǎn)染分兩類：瞬時(shí)轉(zhuǎn)染（ transient transfection）和穩(wěn)定轉(zhuǎn)染（ Stable cell lines） Expression of siRNA