Examining the influence of microRNAs on translation efficiency and on mRNA deadenylation and decay

Abstract

MicroRNAs (miRNAs) utilize multiple posttranscriptional mechanisms to downregulate gene expression in metazoan organisms. These include translation repression and accelerated mRNA decay, the latter being triggered either by deadenylation or, less frequently, by endonucleolytic cleavage, as governed by the degree of complementarity of the targeted message. This chapter describes methods for examining the effect of miRNAs on the translation and turnover of complementary mRNAs in cultured mammalian cells. Among these are procedures for quantifying their influence on the cytoplasmic concentration and translation efficiency of luciferase reporter mRNAs, for monitoring their impact on the deadenylation and decay of beta-globin reporter mRNAs, and for detecting miRNA-directed internal mRNA cleavage.