Poly (a) Enhanced The Effect And Mechanism Of Sirna Gene Silencing Effect Of Exogenous

RNA interference (RNAi) is a powerful tool in the study of gene function. In this study, we report for the first time that poly (A)-tailed siRNAs can nonspecificly enhance the silencing effect on exogenous genes induced by siRNA in MCF-7 cells. Calf intestinal alkaline phosphatase was used to remove 5'-triphosphate groups during the synthesis of des-3'-UU siRNAs against enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP). Poly(A) tails were added to their antisense strands by in vitro transcription, and mechanisms of the silencing effects of poly (A)-tailed siRNAs on EGFP and RFP genes were investigated. Our results showed that siRNAs exhibited single-stranded poly(A) tails on the 3' ends of their antisense strands had stronger silencing effect on exogenous reporter genes than their parental forms. The enhanced silencing effect was non-specific and independent of the siRNA sequence but was related to the length of poly(A). Results also indicated that single strands of poly(A) longer than 50nt could also silence exogenous genes non-specifically, demonstrated that poly(A) is responsible for the enhanced-silencing effect of siRNA-poly(A) on exogenous genes. We found that siRNA-poly(A) could activate PKR and reduce the stability of exogenous-reporter-gene mRNA both in vitro and in vivo, suggesting a possible mechanism of this nonspecific silencing effect on exogenous genes. Our results suggested that poly (A) or noncoding small RNA with poly (A) tail in vivo might be involved in cellular defense against exogenous genes.