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Learn MoreMicroRNAs function is defined by the targets it regulates. Despite miRNAs crucial role in various cellular processes and disease states, the identification of their targets remains challenging. Existing methods suffer from high false-positive rates and are inadequate in differentiating direct targets from downstream regulatory changes. Here, we introduce a simple approach involving combined analysis of RNA-seq and Precise RunOn-seq (PRO-seq) (CARP) to effectively measure post-transcriptional regulation. We apply this approach to seven different miRNAs and robustly distinguish their direct targets from downstream changes. We validate the efficacy of our approach using argonaute-CLIP-seq. We show using CARP and ribosome-profiling analyses that accelerated mRNA decay is the major mode of target regulation by miRNAs. Additionally, our comparative analyses reveal that CARP aids in discovery of complex regulatory mechanisms and in capturing false-negatives of existing approaches. Furthermore, post-transcriptional changes measured using CARP uncover miRNA-dependent regulation of coding sites. Lastly, we demonstrate that PRO-seq provides mechanistic explanation of indirect targeting, unraveling miRNA regulatory networks. Finally, we believe that CARP is particularly suitable to study in vivo functions of miRNAs in primary cells. SOURCE: Andrew Grimson Cornell University
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