RNAiinterference干擾機(jī)制

1、RNA Interference (II)siRNAsiRNAs Small interfering RNAs that have an integral role in the phenomenon of RNA interference(RNAi), a form of post-transcriptional gene silencing RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation A single base

2、pair difference between the siRNA template and the target mRNA is enough to block the process.Mammals exhibit potent responses to dsRNAPKRPPPeiF2a adsRNABlockage of protein synthesisinterferonproductioncell deathapoptosisdsRNA 進(jìn)入哺乳動(dòng)物成體細(xì)胞后，細(xì)胞內(nèi)病毒防御機(jī)制被激活。細(xì)胞內(nèi)干擾素產(chǎn)生增加，蛋白激酶PKR激活，使轉(zhuǎn)錄因子E2F被抑制，非特異性的阻斷基因的轉(zhuǎn)錄，并誘

3、導(dǎo)細(xì)胞凋亡。General mechanism of siRNADICERDouble-stranded RNA triggers processed into siRNAs by enzyme RNAse III family, specifically the Dicer family.Dicer family proteins are ATP-dependent nucleases. These proteins contain an amino-terminal helicase domain, dual RNAseIII domains in the carboxy-terminal

4、 segment, and dsRNA-binding motifsThey can also contain a PAZ domain, which is thought to be important for protein-protein interaction. Dicer homologs exist in many organisms including C. elegans, Drosphila, yeast and humans Loss of dicer: loss of silencing, processing in vitro Developmental consequ

5、ence in Drosophila and C. elegans1. Dicer是是RNaseIIIRNaseIII家族中的一員家族中的一員, ,是特異性識(shí)別雙鏈?zhǔn)翘禺愋宰R(shí)別雙鏈RNARNA的核酸內(nèi)切酶，的核酸內(nèi)切酶，2.2. 以一種以一種ATPATP依賴的方式逐步切割由外源導(dǎo)入或者由依賴的方式逐步切割由外源導(dǎo)入或者由轉(zhuǎn)基因、病毒感染等各種方式引入的雙鏈轉(zhuǎn)基因、病毒感染等各種方式引入的雙鏈RNARNA，3.3. 切割將切割將RNARNA降解為降解為1921bp1921bp的雙鏈的雙鏈RNAs(RNAs(siRNAssiRNAs) )，每個(gè)片斷的每個(gè)片斷的33端都有端都有2 2個(gè)堿基突出。個(gè)堿

6、基突出。 4.4. 該蛋白含有解旋酶（該蛋白含有解旋酶（helicasehelicase），），PAZPAZ，兩個(gè)，兩個(gè)RnaseRnase IIIIII等結(jié)構(gòu)域。等結(jié)構(gòu)域。processing the dsRNA into 21-23 nt fragments3427212016short-interfering RNAQuickTime and aGIF decompressorare needed to see this picture.step one: Tuschl, 2001What cleaves the dsRNA into 21-23 nt fragments?RNase I

7、II enzymes were previously known to cleave both strands of dsRNAs to leave 2 nt 3 overhangsThe same types of products formed when the dsRNA is degradedThe Drosophila genome appears to encode 3 classes of RNase III enzymes1.RNase III - RNC_CAEEL2.Drosha3.2 RNase III domains, 1 helicase domain (CG4792

8、 and CG6493)Are RNase III enzymes involved in siRNA synthesis?(cleavage of ribosomal RNA (rRNA) precursors) Transfect Drosophila S2 cells with T7 epitope tagged-RNase IIIexpression vectorsImmunoprecipitate with T7antibodiesTest whether immunoprecipitatescan generate 21-23 nt fragmentsfrom dsRNACG479

9、2 protein can generate siRNAsCG4792 was renamed Dicer-1CG6493 = Dicer-2Dicer contains two RNAse III domainssiRNAslong dsRNAsiRNAs have a defined structure19 nt duplex2 nt 3 overhangsTuschl, Cell, 2002the antisense strand of the siRNA guides cleavagestep two: RNAi silencing complex (RISC) may be asso

10、ciated with translating ribosomes active RNAse enzyme (Slicer) identified ? may participate in endogenous pathwaysNat Rev Genet 3:737 (2002)“siRNA” molecule:“Dicer” processeslong dsRNA intoshort siRNA:“Guide” (antisense) strand incorporated into RISC complex:guides RISC to complementary sequences in

11、 target mRNAs(RNA-induced silencing complex)Loaded onto Ago2RISC complex RISC is a large (500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNAcleaved mRNA is degraded by cellular exonucleases 現(xiàn)有的現(xiàn)有的RNAiRNAi機(jī)制的模型機(jī)制的模型 通過(guò)生化和遺傳學(xué)研究，產(chǎn)生了現(xiàn)有的通過(guò)生化和遺傳學(xué)研究，產(chǎn)生了現(xiàn)有的RNAiRNAi作用機(jī)制作用

12、機(jī)制模型，包括以下階段：模型，包括以下階段： （1 1）起始階段：）起始階段： 在起始步驟，加入的在起始步驟，加入的dsRNAdsRNA由由DicerDicer切切割為割為21-2321-23核苷酸長(zhǎng)的小分子干擾核苷酸長(zhǎng)的小分子干擾RNARNA片段（片段（small small interfering RNAs interfering RNAs ，siRNAssiRNAs，又被稱為引導(dǎo)，又被稱為引導(dǎo)RNAsRNAs：“guide RNAs” guide RNAs” ）. . （2 2）siRNAsiRNA雙鏈同一些雙鏈同一些RNAiRNAi特異性酶（如特異性酶（如Ago-2)Ago-2)及相關(guān)

13、因及相關(guān)因子共同組成合一個(gè)核酶復(fù)合物，從而形成子共同組成合一個(gè)核酶復(fù)合物，從而形成RNARNA誘導(dǎo)沉默復(fù)誘導(dǎo)沉默復(fù)合物（合物（RNA-induced silencing complex, or RISCRNA-induced silencing complex, or RISC）。）。 （3 3）效應(yīng)階段：激活）效應(yīng)階段：激活RISCRISC需要一個(gè)需要一個(gè)ATPATP依賴的將依賴的將siRNAsiRNA解解雙鏈的過(guò)程。激活的雙鏈的過(guò)程。激活的RISCRISC通過(guò)堿基配對(duì)定位到同源通過(guò)堿基配對(duì)定位到同源mRNAmRNA轉(zhuǎn)轉(zhuǎn)錄本上，并在距離錄本上，并在距離siRNA3siRNA3端端1212個(gè)堿

14、基的位置切割個(gè)堿基的位置切割mRNAmRNA。研究表明每個(gè)研究表明每個(gè)RISCRISC都包含一個(gè)都包含一個(gè)siRNAsiRNA和一個(gè)不同于和一個(gè)不同于DicerDicer的的RNARNA酶。酶。 （4 4）擴(kuò)增階段：）擴(kuò)增階段： 實(shí)驗(yàn)發(fā)現(xiàn)，在新手的實(shí)驗(yàn)發(fā)現(xiàn)，在新手的siRNAsiRNA中，有一部分是通過(guò)一中，有一部分是通過(guò)一種種RNARNA聚合酶的鏈?zhǔn)椒磻?yīng)而產(chǎn)生的。在植物和線蟲(chóng)中，存在聚合酶的鏈?zhǔn)椒磻?yīng)而產(chǎn)生的。在植物和線蟲(chóng)中，存在RNARNA依賴依賴RNARNA聚合酶（聚合酶（RdRpRdRp），參與），參與siRNAsiRNA的擴(kuò)增過(guò)程。的擴(kuò)增過(guò)程。 由于在一些生物中由于在一些生物中RNAi

15、的影響格外顯著，有人提出的影響格外顯著，有人提出在在RNAi途徑中可能存在某個(gè)（信號(hào)）擴(kuò)增的步驟。途徑中可能存在某個(gè)（信號(hào)）擴(kuò)增的步驟。這種擴(kuò)增可能是復(fù)制外源注入的這種擴(kuò)增可能是復(fù)制外源注入的dsRNA從而產(chǎn)生更從而產(chǎn)生更多的多的siRNA，也可能是直接擴(kuò)增，也可能是直接擴(kuò)增siRNA本身。這種本身。這種擴(kuò)增可能在擴(kuò)增可能在RNA誘導(dǎo)沉默復(fù)合物（誘導(dǎo)沉默復(fù)合物（RISC）形成過(guò)程）形成過(guò)程進(jìn)行，作為進(jìn)行，作為RISC形成的補(bǔ)充，或者獨(dú)立于形成的補(bǔ)充，或者獨(dú)立于RISC。 以以SiRNA作為引物，以作為引物，以mRNA為模板，在為模板，在RdRP作作用下合成出用下合成出mRNA的互補(bǔ)鏈。結(jié)果的互

16、補(bǔ)鏈。結(jié)果mRNA也變成了也變成了雙鏈雙鏈RNA，它在，它在Dicer酶的作用下也被裂解成酶的作用下也被裂解成siRNA。這些新生成的這些新生成的siRNA也具有誘發(fā)也具有誘發(fā)RNAi的作用，通過(guò)的作用，通過(guò)這個(gè)聚合酶鏈?zhǔn)椒磻?yīng)，細(xì)胞內(nèi)的這個(gè)聚合酶鏈?zhǔn)椒磻?yīng)，細(xì)胞內(nèi)的siRNA大大增加，大大增加，顯著增加了對(duì)基因表達(dá)的抑制。從顯著增加了對(duì)基因表達(dá)的抑制。從21到到23個(gè)核苷酸個(gè)核苷酸的的siRNA到幾百個(gè)核苷酸的雙鏈到幾百個(gè)核苷酸的雙鏈RNA都能誘發(fā)都能誘發(fā)RNAi，但長(zhǎng)的雙鏈但長(zhǎng)的雙鏈RNA阻斷基因表達(dá)的效果明顯強(qiáng)于短的阻斷基因表達(dá)的效果明顯強(qiáng)于短的雙鏈雙鏈RNA。 siRNA biogenes

17、is Dicer (type III RNAse III) cleaves long dsRNA into siRNA 21-25nt dsRNA from exogenous sources Symmetric 2nt 3 overhangs, 5 phosphate groups Evidence for amplification in C. elegans and plantsRNA Induced Silencing Complex (RISC) RNAi effector complex Preferentially incorporates one strand of unwou

18、nd RNA Khvorova et al., 2003 Antisense How does it know which is which? The strand with less 5 stability usually incorporated into RISC Schwarz et al., 2003Custom-made siRNAssiRNA designMittal, 2004siRNA design 21-23nt 2-nt 3 overhangs ( UU overhangs ) G/C content: 30-50%. No basepair mismatch BLAST

19、 : eliminate any target sequences with significant homology to other coding sequences. design and test 34 siRNA sequences http:/ from plasmidtemplates can inhibit HIV-1Plasmid expression under T7 RNA pol. promoter of self-complementary RNA; results in dsRNA “hairpin” ALL suppressed viral production

20、20-30 xFive Ways to Produce siRNAs In vitro: in vitro preparation of siRNA introduced directly into mammalian cells by transfection, electroporation, or by another method. 1. Chemical synthesis2. In vitro transcription3. Digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNase III)Fi

21、ve Ways to Produce siRNAsIn vivo: the transfection of DNA-based vectors and cassettes that express siRNAs within the cells. 4. Expression in cells from an siRNA expression plasmid or viral vector 5. Expression in cells from a PCR-derived siRNA expression cassetteChemical Synthesis high quality, chem

22、ically synthesized siRNAs on a custom basis. the large yield of high purity siRNA obtained. most expensive Best for: Studies that require large amounts of a defined siRNA sequence Not suitable for: Screening siRNA sequences (cost prohibitive), long term studiesIn Vitro Transcription Relative cost pe

23、r gene: Moderate little hands on time Relative ease of transfection Best for: Screening siRNA sequences or when the price of chemical siRNA synthesis is an obstacle Not suitable for: Long term studies or studies that require large amounts of a single siRNA sequenceA problem In vitro transcription us

24、ing T7 RNA polymerase requires that the first 2 nucleotides of the RNA transcript be GG or GA to ensure efficient synthesis (Milligan 1987). Requiring a GG or GA at the 5 ends of both the sense and antisense strands of an siRNA in addition to the required 3 terminal UU greatly reduces the number of

25、potential target sites for siRNA experiments. This constraint essentially eliminates in vitro transcription as a viable option for preparing siRNAs.Silencer siRNA ConstructionKit -AmbionUse of Chemically Synthesized and in Vitro Transcribed siRNAs to Induce -Actin Gene SilencingHeLa cells siRNA prep

26、aration:chemical synthesis (Ambion) in vitro transcription(Ambions Silencer siRNA Construction Kit)Transfection: siPORT Lipid (Ambion) w/ a 75 nM siRNA3. Digestion of Long dsRNA by an RNase III Family Enzyme long dsRNA: 2001000 nt (in vitro transcription) digest in vitro with RNase III (or Dicer) re

27、move any undigested dsRNA Transfection no need to design and test several siRNA sequences before an effective one can be identifiedthe theoretical potential for nonspecific silencing effectsBest for: Fast and inexpensive analysis of loss of function phenotypesNot suited for: Long term studies or stu

28、dies that require a single, defined siRNA sequence 200 nt of the Ku-70 mRNA HeLa cellsIn Vivo Expression no need to work directly with RNA Expression in cells from an siRNA expression plasmid or viral vector Expression in cells from a PCR-derived siRNA expression cassette4. Expression in cells from

29、an siRNA expression plasmid or viral vectorsiRNA Expression Vectors RNA polymerase III (pol III) : human U6 promoters mouse U6 promoters the human H1 promoter RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs in mammalian cells and

30、 it terminates transcription upon incorporating a string of 36 uridines. pSUPER - Thijn R. Brummelkamp,Ren Bernards,Reuven Agami. A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science, Vol. 296, 550-553, April 19, 2002polymerase-III H1-RNA gene promoter: produces a sma

31、ll RNA transcript lacking a poly-adenosine tail well-defined start of transcriptiona termination signal consisting of five thymidines in a row (T5). the cleavage of the transcript at the termination site is after the second uridine yielding a transcript resembling the ends of synthetic siRNAs, which

32、 also contain two 3 overhanging T or U nucleotides (nt). http:/ Synthesis of two complementary oligonucleotides and hybridizationBoth oligonucleotides are designed such that the first four bases create 5 overhangs compatible with Bbs I (TCCC for the sense strand and AAAC for the antisense strand). I

33、n the sense strand, the 5 overhang is followed by an A (transcription initiation point of the human H1 promoter), then the target sequence of 18-22 mer, 5 to 7 bases for the spacer region, and the inverted 18-22 mer sequence. The sense strand ends with TT to reconstitute the T5 terminator sequence.2

34、. Ligation into psiRNA linearized with Bbs IDigestion with Bbs I liberates the lacZ cassette and creates uncompatible cohesive ends. This increases the number of recombinant clones with an insert in the proper orientation.3. Transformation of E. coli GT116 strainGT116 is an engineered E. coli strain

35、 compatible with hairpin structures.4. DNA extraction and sequencing of the siRNA insert.The psiRNA has been optimized so that analysis of only 5 white transformed colonies is sufficient to obtain the expected siRNA.siRNA Expression Vectors1.more effective than synthetic siRNA 2.Very stable and easy

36、 to handle: 3.Stable cell line can be established: 4.Inducible system can be established: 5.Unlimited supply: once a DNA construct is made, you will have unlimited supply of siRNA. 6.Cost-effective: 7.One big obstacle : it takes a lot of time and trouble to make the DNA constructs. siRNA Expression

37、Vectors long term studies and antibiotic resistance markers Best for: Long term and other studies in which antibiotic selection of siRNA containing cells is desired Not suitable for: Screening siRNA sequences (note: screening siRNA sequences is possible, but is time and labor intensive with vectors)

38、5. siRNA Expression Cassettes (SECs) siRNA Expression Cassettes (SECs) PCR-derived siRNA expression templates PCR product introduced into cells directly without first being cloned into a vector. Castanotto et al., (2002) RNA 8:1454 Rapidly prepare siRNA expression cassettes by PCR no cloning, plasmi

39、d preps or sequencing necessary Quickly test different siRNA sequences and promoters before cloning into a vector Avoid costly siRNA synthesis Variable Reduction in Target Gene Expression Using SECs with Different Promoterspromoters : mouse U6 (Mo-U6), human U6 (Hu-U6), human H1 (Hu-H1) c-fos geneHe

40、La cellsa negative control siRNA (scramble)Length and Sequence of the Loop Linking Sense and Antisense Strands of Hairpin siRNASummaryChemical and IVT synthesis: Allows rapid screening of multiple targets High siRNA homogeneity and purity RNase III mediated hydrolysis: Eliminates the need for screen

41、ing of target site Overcomes variability in silencing by synthetic/IVT siRNA Cost-effective for functional genomic studies PCR based siRNA expression cassettes: Ideal for screening siRNA sequences prior to cloning in a vector Rapid and inexpensive procedure Vector based in vivo expression: Permits l

42、ong-term and stable gene silencing Possibility to use inducible/repressible markers Use of viral vectors Optimize Transfection of siRNAs for RNAi Cell confluence: For most adherent cells, the optimal confluence for transfection is 30-70%. Choice of transfection agent. Determine the optimal volume of transfection agent. Quality and Quantity of siRNA: chemically synthesized siRNA: 1-100 nM in vitro transcribed siRNA: 0.1 to 10 nM for Effect of serum on transfection.Con