The Mechanism Of SiRNA Loading On RISC In The RNA Interference Machinery Of Mammalian Cells

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 Mg²⁺ -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.