哺乳动物细胞RNA干涉过程中siRNA链装载入活性RISC复合体机制的研究
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摘要
RNA干涉(RNA interference,RNAi),是由双链RNA(Double-StrandedRNA简称dsRNA)介导的转录后基因沉默的现象。双链RNA在细胞内被一种称之为Dicer的核酸酶的作用下,被剪切成含3'端两个突出碱基的约21～23nt的小的RNA双链体,称为小干涉RNA(small interfering RNA,siRNA)。siRNA与Dicer等一些蛋白质相互结合,形成RNA诱导的沉默复合物(RISC),根据碱基配对规律特异性识别与之同源的RNA分子,并将其降解。生物学研究发现RNA干涉在抗病毒感染免疫、维持基因组中转座子的稳定性、清除异常RNA和参与基因表达的调控方面发挥重要作用。鉴于其重要的生物学功能,RNA干涉已成为研究基因功能及基因靶向治疗等方面的重要工具。尽管已广为应用,RNA干涉的机制尤其是核心效应复合体RISC的形成过程尚不清楚。
survivin基因是凋亡抑制蛋白家族(IAP)成员之一,其基因定位于第17号染色体,由4个外显子和3个内含子组成。survivin基因编码蛋白为一种癌胚蛋白,在胚胎组织和各种恶性肿瘤组织中均有表达。效应细胞蛋白酶受体1(Effector protease receptor1,EPR1)为凝血蛋白酶因子Xa的受体,其基因亦定位于17号染色体,由1165个碱基组成,编码一337个氨基酸的蛋白。有关其蛋白表达的研究较少,目前仅发现部分血液系统肿瘤中有所表达。survivin基因编码区与EPR1基因序列反向互补。这一特征提示一个共表达survivin和EPR1的细胞将是我们研究哺乳动物细胞RNA干涉过程中siRNA链装载入RISC复合体机制的天然模型。本研究中首先利用Western blot和RT-PCR技术发现HEK-293细胞系共表达survivin和EPR1,为我们的研究奠定了基础。
一系列细胞外共孵育实验结果提示siRNA在进入RISC时,5'端的热力学稳定性较弱的一条链优先进入蛋白复合体,而另外一条链则被抛弃。为了探索细胞内情况是否如此,我们根据热力学稳定性原则,针对survivin和EPR1的互补区设计了6个siRNA,将其转染HEK-293细胞系后,从蛋白水平和mRNA水平检测这两个基因的表达变化,以观察siRNA链作用的情况。结果发现,与对照组相比,各siRNA转染组EPR1和survivin mRNA和蛋白水平均显著降低。
将两条链5'端热力学稳定性相近的siRNA的正义链5'端和3'端第一个碱基进行错配,以观察热力学稳定性对siRNA链作用的影响,结果显示,原siRNA相比,错配siRNA对survivin和EPR1两个基因表达的抑制作用发生同等程度的下降,说明热力学稳定性的变化并没有改变siRNA装配形成RISC时效应链选择的倾向性。
已有研究发现在哺乳动物细胞内Dicer的底物与21nt的siRNA相比,具有更大的作用潜力,提示Dicer的作用可能会促进siRNA链进入到RISC,而且Dicer与siRNA的作用是一种非对称性的酶-底物作用模式。为了探讨Dicer的切割方向是否会影响siRNA链的作用,我们设计了分别针对热力学稳定性相近和差异的siRNA构建了2种发夹状ShRNA表达载体,其转录产物中发夹环分别位于siRNA原序列的3'端和5'端,这种变化使得Dicer的作用方向随着发夹环的位置发生相应的变化。将这些载体转染共表达survivin和EPR1的HEK-293细胞系后,从蛋白水平和mRNA水平检测这两个基因的表达变化,以观察发夹环的位置,即Dicer的作用方向对链选择情况的影响。结果显示与对照组相比,EPR1和survivin mRNA和蛋白水平均显著降低,提示哺乳动物Dicer对发夹环或者长dsRNA的切割方向并不影响链的选择。
有关siRNA双链解链的观点有两种:一种认为RNAi的发动机Ago2将不发挥作用的随从链作为向导链的第一个靶标,将其降解;另外一种观点认为siRNA启动的RISC并不需要ATP的参与,由于已经明确RISC形成过程中链的降解为一种耗能过程,因而推测可能其他机制在此过程发挥作用。哺乳动物细胞内的情况支持哪种观点呢?为了回答这一疑问,我们对siRNA的一条链或者双链的C,U碱基同时进行2'氟修饰。该修饰所产生的空间位阻将阻碍依赖Mg~(2+)的siRNA单链切割机制,因此这种机制有助于探索链的切割是否在细胞内产生含有单链的活性RISC中发挥作用。实验结果显示,与对照组相比,各转染组中EPR1和survivin mRNA水平和蛋白水平均显著降低。其中,siRNA1两条链C,U碱基均进行2'氟修饰后RNAi的效率降低,而仅正义链或者反义链修饰组对目的基因表达的抑制作用几乎不受影响,尤其是单链修饰的siRNA对EPR1和survivin两个基因表达的抑制作用没有显著差异,提示切割可能并不是随从链去除的主要机制。
为了进一步证实RISC复合体形成过程中siRNA两条链是否均有可能进入RISC复合体,我们化学合成了分别与siRNA正义链和反义链互补的生物素化的寡核苷酸。用这种寡核苷酸去捕获转染了相应siRNA的细胞中的RISC复合体,结果发现,无论是在细胞内两条链均可以找到靶标的siRNA,还是只有一条链可以找到靶标的siRNA转染细胞后,分别与其正义链和反义链互补的寡核苷酸链均可以通过碱基配对的方式捕获活性的RISC。
已有研究证实TRBP—Dicer2- Ago2的复合物可以加工pre-miRNA、识别向导链和随从链并且形成成熟的RISC。酵母双杂交实验结果证实TRBP通过第三个双链RNA结合结构域(dsRDB C)与Dicer相互结合。由于Ago2本身不能与siRNA结合,加之以上研究结果提示Dicer也不具有选择siRNA链的能力,那么是否另外一个蛋白TRBP具有传递双链RNA至Ago2的能力?在此过程中,TRBP的dsRBD结构域是否具有链选择的特性?为了明确以上问题,我们构建了一系列可以表达TRBP结构域的pFLAG-CMV4载体,将其与siRNA一同转染HEK-293细胞后,通过捕获RISC来观察TRBP的dsRBD结构域在RISC形成过程中的作用。结果我们发现任一dsRBD结构域的缺失并不影响活性RISC复合体的形成。
综上所述,本研究应用一个天然的细胞模型,通过改变siRNA链的特性,研究两条链分别作为效应链抑制相应基因表达的能力,证实在哺乳动物细胞中siRNA双链均可以与相关蛋白结合形成活性RISC复合体,而且这种特性不受热力学稳定性因素以及Dicer作用方向的影响。同时,研究结果提示RISC复合体中siRNA的解链并不是通过切割随从单链的方式发挥作用。本研究从细胞天然模型这一新的视角入手,为阐明哺乳动物细胞中RNA干涉的详细机制提供了新的实验依据。
RNA interference(RNAi) is a gene-silencing process during which endogenous messenger RNA is destroyed upon introduction of the corresponding double-stranded RNA.dsRNA is processed by the cellular enzyme Dicer into short,21～23 nucleotide dsRNA segments,referred to as small interfering RNA(siRNA),with a two-nucleotide overhang at each 3' end.Theses siRNA become incorporated into a RNA-induced silecing complex(RISC),where siRNA serves as a guide to identify homologous mRNA for destruction. Biologically,RNAi-related processes are critical for development, heterochromatin formation,and offer cellular protection from virus and transposon proliferation.RNAi has found widespread application as a technique within research laboratories,allowing simple yet effective knockdown of genes of interest.Despite the widespread use of RNAi to knockdown gene function,the RNAi pathway itself remains poorly understood,especially for the formation of RISC complex.
Survivin,is a member of IAP members which are the important gene families responsible for apoptosis regulation.Survivin gene is localized at chromosome 17 and has 4 exons and 3 introns.The onco-fetal survivin protein is expressed in embryonic organisms and in various malignant tumors.Localized on chromosome 7,the effector protease receptor1(EPR1) gene encodes a protein with 337 amino acids.The EPR-1 mRNA contains a 1011 nt region complementary to survivin mRNA with only 5nt variantion and 6nt insertion, which thus provided a natural model for exploring the functions of siRNA duplex and the formation of RISC in culture cells.As an initial test of our idea,EPR1 and survivin in several cell lines was screened using Western blot,and HEK-293 cells was selected as a cell line expressing both genes simultaneously.
Several in vitro observations have suggested that thermodynamic asymmetry governs strand selection for processed RNAi triggering,with only guide strand of siRNA duplex is incorporated in the RISC.To assess if only the guide strand of siRNA duplex is competent to induce RNAi and if the selection of the guide strand always abides by the thermodynamic asymmetry rule,we designed 6 siRNA duplexes,with different thermodynamic profiles at either the 5' or the 3' end,toward the complementary region of EPR1 and survivin.After being transfected into HEK-293 cells,the inhibited expressions of both EPR1 and survivin were observed in our experiment for siRNA duplexes with different thermodynamic profile.To further verify the influence of thermodynamic profiles of siRNA duplexes to the selection of guide strand,we synthesized two kind of siRNA duplexs,one of the siRNA containing a 5'end mismatched base pair corresponding to siRNA1 and the other containing a 3' end mismatch.Our data showed that the mismatchs in siRNA duplexs resulted in comparably suppressed knockdown of EPR1 and survivin,indicating that changing the thermodynamic profiles of the siRNA duplex cannot influence the selection of the guide strand.
In mammalian cells,Dcr substrates(such as short hairpin RNAs or 27 nt duplexes) exhibit greater silencing potency than 21 nt siRNAs,suggesting that Dcr processing might facilitate entry into the RISC assembly pathway.During dsRNA processing,Dicer liberates siRNA from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions.To test whether Dcr processing polarity influences siRNA strand selection,we constructed short hairpin RNA(shRNA) vectors that transcribe shRNAs in which the stem loop locats at 3' and 5' ends of the siRNA.With similar thermodynamic profiles individually, these shRNAs permit processing specifically from either the 5' or the 3' end of the incipient siRNA.We transfected the shRNA vectors into HEK-293 cells and examined the expression of EPR1 and survivin by RT-PCR and Western blot.In all cases,shRNA-R and shRNA-L,which contain a loop at the sites corresponding to the 3' and 5' end of the sense strand of the original siRNA, respectively,triggered similarly efficient RNAi and the cleavage of their corresponding mRNA targets.We concluded that the direction of Dcr processing is not likely to influence selection of guide strand in mammalian cells.
There are two established concerns about the unwinding of siRNA duplex now.For one point,Ago2 cleaves the passenger strand during the RISC conformation.For the other,due to no ATP incorporating in the initiation the of siRNA loading on the RISC,it can not provide the energy for passenger strand cleavage.Therefore,maybe there are alternative solutions for removing certain anti-guide siRNA strands.To explore this question,we incorporated the 2'FC,FU modification on the sense strand and/or antisense strand of siRNA1 duplexes. This modification can be used to specifically block ribonuclease activity including RISC activity toward RNA targets.The inhibitive effect is due to the ability of the Fluro group to cause steric hindrance of the Mg~(2+) -dependent cleavage of the passenger strand.We then examined the levels of EPR1 and survivin following transfection of HEK-293 cells with the modified siRNA duplex.Our data showed that the 2'-fluorination modification of Cytosines and Uracils on the sense or antisense strand of siRNA didn't alter the efficacy and potency of either EPR1 or survivin silencing.Whereas both the silencing of EPR1 and survivin were significantly inhibited by a concurrent modification of both strands,the knockdown of both genes still can be observed.It seems to us that the cleavage event is not critical for the passenger strand to leave the RISC and therefore activate the cleavage of mRNA substrate.Modification of both strands may hinder the unwinding of duplex and then influence the RNAi activity via interfering with the binding of proteins involved in the RNAi process.
To further ask whether both strands of siRNA can be incorporating into the active RISC in mammalian cells.We used 2'FU,FC oligonucleotides,which were tethered to streptavidin paramagnetic beads via a 5' biotin linkage and complementary to the sense and antisense strands of synthetic siRNAs,to capture the siRNA-programmed RISC in vivo.Our results showed that both 2'FU,FC oligonucleotides complementary to the sense and antisense strands of siRNA can capture active RISC in vivo.
It is confirmed that TRBP-Dicer2-Ago2 complex can process the pre-miRNA and recognize the passenger strand and guide strand during the activation of RISC.Dicer and TRBP interact in a yeast two-hybrid(2H) assay through the carboxy-terminal domain of TRBP(dsRBD C).Argonaute2(Ago2) cannot associated with siRNA complex on its own,and analysis of the interaction between the TRBP,Ago2 and siRNA indicated that TRBP may have a role in mediating Dicer association with siRNA and the recruitment of Ago2.To better understand which double-stranded RNA binding domains(dsRBDs) of TRBP is responsible for the delivery of siRNA to Ago2,we co-transfected siRNAs and serially truncated TRBP containing Flag-tagged single or tandemed different dsRBDs.We found that the deletion of any of the single dsRBDs does not influence the formation and activation of RISC.
In summary,we studied the mechanism underlying siRNA strand selection in triggering target gene silencing in a natural cellular model via altering the related parameters of siRNAs,and confirmed that both strands of siRNA duplex can be incorporated into the active RISC.The thermodynamic parameters and Dicer processing polarity do not alter this property.In addition,our results indicate that the leave of passenger strand is not dependent on the cleavage mechanism.Our study provides new insights into the detailed mechanisms of RNA interference in mammalian cells.
survivin基因是凋亡抑制蛋白家族(IAP)成员之一,其基因定位于第17号染色体,由4个外显子和3个内含子组成。survivin基因编码蛋白为一种癌胚蛋白,在胚胎组织和各种恶性肿瘤组织中均有表达。效应细胞蛋白酶受体1(Effector protease receptor1,EPR1)为凝血蛋白酶因子Xa的受体,其基因亦定位于17号染色体,由1165个碱基组成,编码一337个氨基酸的蛋白。有关其蛋白表达的研究较少,目前仅发现部分血液系统肿瘤中有所表达。survivin基因编码区与EPR1基因序列反向互补。这一特征提示一个共表达survivin和EPR1的细胞将是我们研究哺乳动物细胞RNA干涉过程中siRNA链装载入RISC复合体机制的天然模型。本研究中首先利用Western blot和RT-PCR技术发现HEK-293细胞系共表达survivin和EPR1,为我们的研究奠定了基础。
一系列细胞外共孵育实验结果提示siRNA在进入RISC时,5'端的热力学稳定性较弱的一条链优先进入蛋白复合体,而另外一条链则被抛弃。为了探索细胞内情况是否如此,我们根据热力学稳定性原则,针对survivin和EPR1的互补区设计了6个siRNA,将其转染HEK-293细胞系后,从蛋白水平和mRNA水平检测这两个基因的表达变化,以观察siRNA链作用的情况。结果发现,与对照组相比,各siRNA转染组EPR1和survivin mRNA和蛋白水平均显著降低。
将两条链5'端热力学稳定性相近的siRNA的正义链5'端和3'端第一个碱基进行错配,以观察热力学稳定性对siRNA链作用的影响,结果显示,原siRNA相比,错配siRNA对survivin和EPR1两个基因表达的抑制作用发生同等程度的下降,说明热力学稳定性的变化并没有改变siRNA装配形成RISC时效应链选择的倾向性。
已有研究发现在哺乳动物细胞内Dicer的底物与21nt的siRNA相比,具有更大的作用潜力,提示Dicer的作用可能会促进siRNA链进入到RISC,而且Dicer与siRNA的作用是一种非对称性的酶-底物作用模式。为了探讨Dicer的切割方向是否会影响siRNA链的作用,我们设计了分别针对热力学稳定性相近和差异的siRNA构建了2种发夹状ShRNA表达载体,其转录产物中发夹环分别位于siRNA原序列的3'端和5'端,这种变化使得Dicer的作用方向随着发夹环的位置发生相应的变化。将这些载体转染共表达survivin和EPR1的HEK-293细胞系后,从蛋白水平和mRNA水平检测这两个基因的表达变化,以观察发夹环的位置,即Dicer的作用方向对链选择情况的影响。结果显示与对照组相比,EPR1和survivin mRNA和蛋白水平均显著降低,提示哺乳动物Dicer对发夹环或者长dsRNA的切割方向并不影响链的选择。
有关siRNA双链解链的观点有两种:一种认为RNAi的发动机Ago2将不发挥作用的随从链作为向导链的第一个靶标,将其降解;另外一种观点认为siRNA启动的RISC并不需要ATP的参与,由于已经明确RISC形成过程中链的降解为一种耗能过程,因而推测可能其他机制在此过程发挥作用。哺乳动物细胞内的情况支持哪种观点呢?为了回答这一疑问,我们对siRNA的一条链或者双链的C,U碱基同时进行2'氟修饰。该修饰所产生的空间位阻将阻碍依赖Mg~(2+)的siRNA单链切割机制,因此这种机制有助于探索链的切割是否在细胞内产生含有单链的活性RISC中发挥作用。实验结果显示,与对照组相比,各转染组中EPR1和survivin mRNA水平和蛋白水平均显著降低。其中,siRNA1两条链C,U碱基均进行2'氟修饰后RNAi的效率降低,而仅正义链或者反义链修饰组对目的基因表达的抑制作用几乎不受影响,尤其是单链修饰的siRNA对EPR1和survivin两个基因表达的抑制作用没有显著差异,提示切割可能并不是随从链去除的主要机制。
为了进一步证实RISC复合体形成过程中siRNA两条链是否均有可能进入RISC复合体,我们化学合成了分别与siRNA正义链和反义链互补的生物素化的寡核苷酸。用这种寡核苷酸去捕获转染了相应siRNA的细胞中的RISC复合体,结果发现,无论是在细胞内两条链均可以找到靶标的siRNA,还是只有一条链可以找到靶标的siRNA转染细胞后,分别与其正义链和反义链互补的寡核苷酸链均可以通过碱基配对的方式捕获活性的RISC。
已有研究证实TRBP—Dicer2- Ago2的复合物可以加工pre-miRNA、识别向导链和随从链并且形成成熟的RISC。酵母双杂交实验结果证实TRBP通过第三个双链RNA结合结构域(dsRDB C)与Dicer相互结合。由于Ago2本身不能与siRNA结合,加之以上研究结果提示Dicer也不具有选择siRNA链的能力,那么是否另外一个蛋白TRBP具有传递双链RNA至Ago2的能力?在此过程中,TRBP的dsRBD结构域是否具有链选择的特性?为了明确以上问题,我们构建了一系列可以表达TRBP结构域的pFLAG-CMV4载体,将其与siRNA一同转染HEK-293细胞后,通过捕获RISC来观察TRBP的dsRBD结构域在RISC形成过程中的作用。结果我们发现任一dsRBD结构域的缺失并不影响活性RISC复合体的形成。
综上所述,本研究应用一个天然的细胞模型,通过改变siRNA链的特性,研究两条链分别作为效应链抑制相应基因表达的能力,证实在哺乳动物细胞中siRNA双链均可以与相关蛋白结合形成活性RISC复合体,而且这种特性不受热力学稳定性因素以及Dicer作用方向的影响。同时,研究结果提示RISC复合体中siRNA的解链并不是通过切割随从单链的方式发挥作用。本研究从细胞天然模型这一新的视角入手,为阐明哺乳动物细胞中RNA干涉的详细机制提供了新的实验依据。
RNA interference(RNAi) is a gene-silencing process during which endogenous messenger RNA is destroyed upon introduction of the corresponding double-stranded RNA.dsRNA is processed by the cellular enzyme Dicer into short,21～23 nucleotide dsRNA segments,referred to as small interfering RNA(siRNA),with a two-nucleotide overhang at each 3' end.Theses siRNA become incorporated into a RNA-induced silecing complex(RISC),where siRNA serves as a guide to identify homologous mRNA for destruction. Biologically,RNAi-related processes are critical for development, heterochromatin formation,and offer cellular protection from virus and transposon proliferation.RNAi has found widespread application as a technique within research laboratories,allowing simple yet effective knockdown of genes of interest.Despite the widespread use of RNAi to knockdown gene function,the RNAi pathway itself remains poorly understood,especially for the formation of RISC complex.
Survivin,is a member of IAP members which are the important gene families responsible for apoptosis regulation.Survivin gene is localized at chromosome 17 and has 4 exons and 3 introns.The onco-fetal survivin protein is expressed in embryonic organisms and in various malignant tumors.Localized on chromosome 7,the effector protease receptor1(EPR1) gene encodes a protein with 337 amino acids.The EPR-1 mRNA contains a 1011 nt region complementary to survivin mRNA with only 5nt variantion and 6nt insertion, which thus provided a natural model for exploring the functions of siRNA duplex and the formation of RISC in culture cells.As an initial test of our idea,EPR1 and survivin in several cell lines was screened using Western blot,and HEK-293 cells was selected as a cell line expressing both genes simultaneously.
Several in vitro observations have suggested that thermodynamic asymmetry governs strand selection for processed RNAi triggering,with only guide strand of siRNA duplex is incorporated in the RISC.To assess if only the guide strand of siRNA duplex is competent to induce RNAi and if the selection of the guide strand always abides by the thermodynamic asymmetry rule,we designed 6 siRNA duplexes,with different thermodynamic profiles at either the 5' or the 3' end,toward the complementary region of EPR1 and survivin.After being transfected into HEK-293 cells,the inhibited expressions of both EPR1 and survivin were observed in our experiment for siRNA duplexes with different thermodynamic profile.To further verify the influence of thermodynamic profiles of siRNA duplexes to the selection of guide strand,we synthesized two kind of siRNA duplexs,one of the siRNA containing a 5'end mismatched base pair corresponding to siRNA1 and the other containing a 3' end mismatch.Our data showed that the mismatchs in siRNA duplexs resulted in comparably suppressed knockdown of EPR1 and survivin,indicating that changing the thermodynamic profiles of the siRNA duplex cannot influence the selection of the guide strand.
In mammalian cells,Dcr substrates(such as short hairpin RNAs or 27 nt duplexes) exhibit greater silencing potency than 21 nt siRNAs,suggesting that Dcr processing might facilitate entry into the RISC assembly pathway.During dsRNA processing,Dicer liberates siRNA from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions.To test whether Dcr processing polarity influences siRNA strand selection,we constructed short hairpin RNA(shRNA) vectors that transcribe shRNAs in which the stem loop locats at 3' and 5' ends of the siRNA.With similar thermodynamic profiles individually, these shRNAs permit processing specifically from either the 5' or the 3' end of the incipient siRNA.We transfected the shRNA vectors into HEK-293 cells and examined the expression of EPR1 and survivin by RT-PCR and Western blot.In all cases,shRNA-R and shRNA-L,which contain a loop at the sites corresponding to the 3' and 5' end of the sense strand of the original siRNA, respectively,triggered similarly efficient RNAi and the cleavage of their corresponding mRNA targets.We concluded that the direction of Dcr processing is not likely to influence selection of guide strand in mammalian cells.
There are two established concerns about the unwinding of siRNA duplex now.For one point,Ago2 cleaves the passenger strand during the RISC conformation.For the other,due to no ATP incorporating in the initiation the of siRNA loading on the RISC,it can not provide the energy for passenger strand cleavage.Therefore,maybe there are alternative solutions for removing certain anti-guide siRNA strands.To explore this question,we incorporated the 2'FC,FU modification on the sense strand and/or antisense strand of siRNA1 duplexes. This modification can be used to specifically block ribonuclease activity including RISC activity toward RNA targets.The inhibitive effect is due to the ability of the Fluro group to cause steric hindrance of the Mg~(2+) -dependent cleavage of the passenger strand.We then examined the levels of EPR1 and survivin following transfection of HEK-293 cells with the modified siRNA duplex.Our data showed that the 2'-fluorination modification of Cytosines and Uracils on the sense or antisense strand of siRNA didn't alter the efficacy and potency of either EPR1 or survivin silencing.Whereas both the silencing of EPR1 and survivin were significantly inhibited by a concurrent modification of both strands,the knockdown of both genes still can be observed.It seems to us that the cleavage event is not critical for the passenger strand to leave the RISC and therefore activate the cleavage of mRNA substrate.Modification of both strands may hinder the unwinding of duplex and then influence the RNAi activity via interfering with the binding of proteins involved in the RNAi process.
To further ask whether both strands of siRNA can be incorporating into the active RISC in mammalian cells.We used 2'FU,FC oligonucleotides,which were tethered to streptavidin paramagnetic beads via a 5' biotin linkage and complementary to the sense and antisense strands of synthetic siRNAs,to capture the siRNA-programmed RISC in vivo.Our results showed that both 2'FU,FC oligonucleotides complementary to the sense and antisense strands of siRNA can capture active RISC in vivo.
It is confirmed that TRBP-Dicer2-Ago2 complex can process the pre-miRNA and recognize the passenger strand and guide strand during the activation of RISC.Dicer and TRBP interact in a yeast two-hybrid(2H) assay through the carboxy-terminal domain of TRBP(dsRBD C).Argonaute2(Ago2) cannot associated with siRNA complex on its own,and analysis of the interaction between the TRBP,Ago2 and siRNA indicated that TRBP may have a role in mediating Dicer association with siRNA and the recruitment of Ago2.To better understand which double-stranded RNA binding domains(dsRBDs) of TRBP is responsible for the delivery of siRNA to Ago2,we co-transfected siRNAs and serially truncated TRBP containing Flag-tagged single or tandemed different dsRBDs.We found that the deletion of any of the single dsRBDs does not influence the formation and activation of RISC.
In summary,we studied the mechanism underlying siRNA strand selection in triggering target gene silencing in a natural cellular model via altering the related parameters of siRNAs,and confirmed that both strands of siRNA duplex can be incorporated into the active RISC.The thermodynamic parameters and Dicer processing polarity do not alter this property.In addition,our results indicate that the leave of passenger strand is not dependent on the cleavage mechanism.Our study provides new insights into the detailed mechanisms of RNA interference in mammalian cells.
引文
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