分子生物學課件分子生物

1、9 Regulatory Small RNAMany eukaryotes share an additional, highly abundant class of small RNAs that range in size from 19 to 28 base. They participate in gene silencing through RNA-RNA and possibly also RNA-DNA interactions, mediate a wide range of phenomena , such as transcriptional silencing of he

2、terochromain, pos-transcriptional regulation of mRNA stability(mRNA cleavage), as well as destruction and silencing of invading viral genome or transgene. These small RNAs (sRNAs) have been found to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat

3、viruses small in eukaryotes. sRNAs are classified into three major class: short interfering RNAs (siRNAs, 20-24nt): heterochromatic siRNAs, ta-siRNAs, natural antisense transcript siRNAs.microRNAs (miRNAs, 21-24nt),Piwi-interacting RNAs (piRNAs, 26-31nt) in metazoans. Major classes of sRNAGene silen

4、cing（also called RNA silencing）, including transcriptional gene silencing （轉(zhuǎn)錄水平基因沉默）和post-transcriptional gene silencing （轉(zhuǎn)錄后基因沉默）。RNA干擾（RNA interference, RNAi）是指由雙鏈RNA誘發(fā)的、同源mRNA高效特異性降解技術(shù)。DNA甲基化組蛋白修飾RNAi介導的gene siliencing 轉(zhuǎn)基因相關(guān)的轉(zhuǎn)錄后沉默(PTGS)及病毒誘導的沉默(VIGS); Tra-siRNAs and miRNA對植物發(fā)育的調(diào)控; 轉(zhuǎn)基因相關(guān)的轉(zhuǎn)錄水平的沉默，

5、即RNA指導的DNA甲基化和異染色質(zhì)的形成。真核生物基因表達的表觀遺傳學調(diào)控9.1 siRNAsiRNA的發(fā)現(xiàn) 1988年，Rich Jorgensen和同事在對矮牽牛（petunias）進行的研究中有個奇怪的發(fā)現(xiàn)：將一個能產(chǎn)生色素的基因置于一個強啟動子后，導入矮腳牽牛中，試圖加深花朵的紫顏色，結(jié)果沒看到期待中的深紫色花朵，多數(shù)花成了花斑的甚至白的。Jorgensen將將這種現(xiàn)象命名為協(xié)同抑制“cosuppression”，因為導入的基因和其相似的內(nèi)源基因同時都被抑制。后來發(fā)現(xiàn)在其他許多植物中，甚至在真菌中也有類似的現(xiàn)象。野生型試驗預測 Effects ofmex-3RNA interfere

6、nce on levels of the endogenous mRNA. Nomarski DIC micrographs showin situhybridization of 4-cell stage embryos. (A) Negative control showing lack of staining in the absence of the hybridization probe. (B) Embryo from uninjected parent showing normal pattern of endogenousmex-3RNA (purple staining).

7、(C) Embryo from parent injected with purifiedmex-3antisense RNA. These embryos (and the parent animals) retainmex-3mRNA, although levels may be somewhat less than wild type. (D) Late 4-cell stage embryo from a parent injected with dsRNA corresponding tomex-3; nomex-3RNA is detected. (Templates used

8、for interfering RNA andin situprobes were largely non-overlapping.)Each embryo is approximately 50 m in length.(For details see:Fireet al.1998 Potent and specific genetic interference by double-stranded RNA inCaenorhabditis elegansNature 391: 806-11)2. siRNA的生物合成Dicer切割形成雙鏈小片段 組裝復合物形成有活性的沉默復合體RISC引導

9、鏈乘客鏈Dicer 是一種 RNAase llI-like enzyme 能夠識別和消化長片段dsRNA；其產(chǎn)物為約23nt的短的雙鏈 dsRNA (通常被稱為短干涉RNAs, 或 siRNAs)；而且每個siRNA 的3 端帶有兩個突出堿基。3. siRNA介導的沉默機理已知siRNA通過以下方式抑制同源基因的表達： 轉(zhuǎn)錄水平的基因沉默，即RNA指導的DNA甲基化和 異染色質(zhì)的形成 轉(zhuǎn)錄后基因沉默，即同源mRNA的降解或抑制mRNA翻譯。Int J Biol Sci2009; 5(2):97-117.近來發(fā)現(xiàn)在裂殖酵母的著絲粒區(qū)域的沉默是通過RNAi machinery. 其中，著絲粒區(qū)域轉(zhuǎn)

10、錄生成的RNAs折疊成莖環(huán)，或者與同一區(qū)域的其它雜交；由此形成的 dsRNAs 可被 Dicer識別，并切割產(chǎn)生小的siRNAs，其主要引導RNAi machinery到著絲粒。RNAi 沉默的另一個值得注意的特征是高效； 因此，極少量的dsRNA 足以使目標基因徹底關(guān)閉.尚不清楚為何會產(chǎn)生如此強的效果，這可能涉及到一種在許多RNAi 實例中都必須的RNA依賴性RNA聚合酶（RNA-dependent RNA polymerase，簡稱RdRP）當原有的siRNA被募集到 mRNA， RdRP 的作用可能使產(chǎn)生更多的siRNAs，而它們將同段mRNA臨近區(qū)作為目標（圖17-30）。4. siR

11、NA的生物學意義在轉(zhuǎn)錄水平、轉(zhuǎn)錄后水平參與基因的表達調(diào)控；維持基因組的穩(wěn)定；保護基因組免受外源核酸侵入。宿主可以以病毒DNA為模板合成雙鏈RNA，從而抑制病毒對宿主的破壞。形成的siRNA還可以通過胞間連絲在細胞間傳遞，讓這些細胞提前具有抵抗病毒的能力。另一方面，病毒也進化出與宿主抗病基因序列相似的RNA，用同樣的機制增加未被侵染細胞的易感性。 Scoeld S. R. et al. Plant Physiology. 2005 BSMV -induce gene silence in wheat spikes/grainsFig. Silencing endogenous phytoene

12、desaturase gene (PDS) expression in wheat spikes/seeds. We demonstrated the feasibility of using BSMV for gene silencing in wheat spikes or grains. .Fig. 4 The protein and mRNA levels of 1Bx14 in the grains of BSMV:1Bx14 infected spikes1.6. Application of the BSMV-VIGS approach to functional analysi

13、s of HMW-GS-encoding genes These results are consistent with our previous study, suggesting the usefulness of the BSMV-VIGS system for functional analysis of genes expressed in wheat grains. Ma Meng et al. BMC Plant Biology. 2012,12:141Fig. Silencing of TaCYP78A3 reduces seed and embryo size in whea

14、t. Fig. 7 Overexpression of TaCYP78A3 can increases organ size and final seed sizeThese results suggested that the TaCYP78A3 gene have functions in affecting seed size 9.2 miRNAmiRNA的發(fā)現(xiàn) miRNA(microRNA)首先中線蟲的lin-4基因中發(fā)現(xiàn)：a| The precursor structure and mature microRNA (miRNA) sequence oflin-4.b| Sequenc

15、e complementarity betweenlin-4(red) and the 3-untranslated region (UTR) oflin-14mRNA (blue).lin-4is partially complementary to 7 sites in thelin-143 UTR; its binding to these sites of complementarity brings about repression of LIN-14 protein synthesis13,18. RISC, RNA-induced silencing complex.Nature

16、 Reviews Genetics5,522-531(July 2004)miRNAs and larval development inCaenorhabditis elegans.Genetic screens identified an miRNAlin-4, which regulates the expression oflin-14by binding tolin-14and inhibiting translation. A second miRNA,let-7, was discovered, which is involved in the same developmenta

17、l timing pathway aslin-4, but is induced at a different stage.lin-4is expressed at the L1 stage, whereaslet-7is expressed during the L3L4 stage.lin-4andlet-7 are induced at distinct times duringC. eleganslarval development to trigger transitions from one larval stage to the next by negatively regula

18、ting the expression of the protein-coding geneslin-14andlin-41, respectively. 通過lin-4、let-7的調(diào)控，實現(xiàn)了線蟲發(fā)育各階段關(guān)鍵基因的正確時空表達調(diào)節(jié)。2. miRNA的生物合成In the standard microRNA (miRNA) biogenesis pathway, primary miRNA (pri-miRNA) transcripts are processed by RNAase Drosha in the nucleus and by Dicer in the cytoplasm.

19、The pri-miRNA, which is transcribed by RNA polymerase II (Pol II), begins with a 7-methylguanosine cap (m7Gppp) and ends with a 3 poly(A) tail. The pri-miRNA contains a stemloop structure that is cleaved in the nucleus by the endonuclease Drosha together with its double-stranded RNA (dsRNA)-binding

20、protein parner DGCR8 (in mammals) or Pasha (in flies). Nature Reviews Molecular Cell Biology14,475488(2013)The resulting precursor miRNA (pre-miRNA) is exported from the nucleus by exportin 5 and then further cleaved by the endonuclease Dicer together with its dsRNA-binding partner TRBP (transactiva

21、tion-response RNA-binding protein; in mammals) or Loquacious (Loqs; in flies) to liberate a miRNAmiRNA*duplex. Supported by the HSC70HSP90 chaperone machinery, this duplex is loaded into an Argonaute (AGO) protein as a dsRNA. Subsequent maturation steps expel the miRNA*, producing a mature RNA-induc

22、ed silencing complex (RISC). Alternative pathways (shown on the right) typically replace individual steps of miRNA precursor processing. Nature Reviews Molecular Cell Biology14,475488(2013)miRNAs, 典型的長度為21 or 22 nts, 產(chǎn)生于非蛋白編碼基因轉(zhuǎn)錄的前體（約 70-90 nts長) 。這些轉(zhuǎn)錄產(chǎn)物含有能形成莖環(huán)結(jié)構(gòu)（stem loop structures）的序列，這種結(jié)構(gòu)由Dicer

23、(or DCL1, for Dicer-like I in plants)加工而成 。通過轉(zhuǎn)錄本產(chǎn)生的 miRNAs 導致體內(nèi)與其同源的靶標mRNAs破壞 (在植物中) or 抑制翻譯 (在線蟲中)。3. Small RNA的選擇性裝配4. miRNA的功能1) 和siRNA一樣裝載成RISC后, 使互補配對的mRNA降解。2) miRNA可抑制mRNA的翻譯，降低靶基因的蛋白質(zhì)水平但 不影響其RNA水平。相同點：1. 二者的長度都約在22nt左右。2. 二者都依賴Dicer酶的加工，是Dicer的產(chǎn)物，所以具有Dicer產(chǎn)物的特點。3. 二者生成都需要Argonaute家族蛋白存在。二者都是

24、RISC組分，所以其功能界限變得不清晰，如二者在介導沉默機制上有重疊。5. miRNA和siRNA合成都是由雙鏈的RNA或RNA前體形成的。5. miRNA與siRNA異同不同點：1. 根本區(qū)別是miRNA是內(nèi)源的，是生物體的固有因素；而siRNA是人工體外合成的，通過轉(zhuǎn)染進入人體內(nèi)，是RNA干涉的中間產(chǎn)物。2. 結(jié)構(gòu)上，miRNA是單鏈RNA，而siRNA是雙鏈RNA。3. Dicer酶對二者的加工過程不同，miRNA是不對稱加工，miRNA僅是剪切pre-miRNA的一個側(cè)臂，其他部分降解；而siRNA對稱地來源于雙鏈RNA的前體的兩側(cè)臂。4. 在作用位置上，miRNA主要作用于靶標基因3

25、-UTR區(qū)，而siRNA可作用于mRNA的任何部位。5. 在作用方式上，miRNA可抑制靶標基因的翻譯，也可以導致靶標基因降解，而siRNA只能導致靶標基因的降解。6. miRNA主要在發(fā)育過程中起作用，調(diào)節(jié)內(nèi)源基因表達，而siRNA不參與生物生長，是RNAi的產(chǎn)物，原始作用是抑制轉(zhuǎn)座子活性和病毒感染。miRNA與siRNA異同6. MicroRNAs 調(diào)控一些發(fā)育期基因的表達MicroRNAs 是很短的RNAs （miRNA），它們可以調(diào)節(jié)基因的表達。動、植物基因組編碼很多mi RNAs(22 base)分子。 MicroRNAs 通過其與目標mRNAs 互補序列配對調(diào)節(jié)基因的表達。據(jù)估計在線蟲中編碼miRNA的基因大概有 120個 ，而在人類有250個.這些miRNA經(jīng)常在發(fā)育調(diào)節(jié)模式中被表達，且它們的靶標是那些編碼在生物發(fā)育上具有重要作用的調(diào)節(jié)蛋白的mRNA圖3. Fig3. Interplay of SPL genes and miRNAs in Arabidopsis development. (Chen X.B. et al., 2012)擬南芥的生命周期分為三個階段：幼年期，成年期和生殖期。不同時期SPL基因和miRNA及其他基因的作用如圖3所示.在