Target site effects in the RNA interference and microRNA pathways

Abstract

In RNAi (RNA interference), siRNAs (small interfering RNAs) are loaded into the RISC (RNA-induced silencing complex), which then mediates endonucleolytic cleavage of complementary target RNAs. Although RNAi has become one of the most powerful tools in molecular biology to assess gene function, there remains a great number of ineffective siRNAs. It is already known that the assembly and activation of RISC is a crucial determinant of RNAi activity, but downstream effects such as target accessibility have not been analysed extensively. Therefore we assessed the effect of target site accessibility and found that it significantly improves the potency of siRNAs. Similarly, miRNAs (microRNAs) act by repressing protein synthesis through imperfect base-pairing to the 3′-UTR (untranslated region) of target mRNAs. We found that predicted target sites reside in regions of high accessibility and tested whether this criterion could be used in the search of functional miRNA targets. In addition, we performed reporter gene assays to test whether accessibility correlates with measured mRNA suppression levels. The results of our initial study suggest that secondary structures might add a so far underrepresented layer of complexity in the recognition of RNA targets by miRNAs.