Integrated analysis of directly captured microRNA targets reveals the impact of microRNAs on mammalian transcriptome

Abstract

MicroRNA (miRNA)-mediated regulation is widespread, relatively mild but functionally important. Despite extensive efforts to identify miRNA targets, it remains unclear how miRNAs bind to mRNA targets globally and how changes in miRNA levels affects the transcriptome. Here we apply an optimized method for simultaneously capturing miRNA and targeted RNA sites to wildtype, miRNA knockout and induced epithelial cells. We find that abundantly expressed miRNAs can bind to thousands of different transcripts and many different miRNAs can regulate the same gene. Although mRNA sites that are bound by miRNAs and also contain matches to seed sequences confer the strongest regulation, [~]50% of miRNAs bind to RNA regions without seed matches. In general, these bindings have little impact on mRNA levels and reflect a scanning activity of miRNAs. In addition, different miRNAs have different preferences to seed matches and 3end base-pairing. For a single miRNA, the effectiveness of mRNA regulation is highly correlated with the number of captured miRNA:RNA fragments. Notably, elevated miRNA expression effectively represses existing targets with little impact on newly recognized targets. Global analysis of directly captured mRNA targets reveals pathways that are involved in cancer, cell adhesion and signaling pathways are highly regulated by many different miRNAs in epithelial cells. Comparison between experimentally captured and TargetScan predicted targets indicates that our approach is more effective to identify bona fide targets by reducing false positive and negative predictions. This study reveals the global binding landscape and impact of miRNAs on mammalian transcriptome.