UC Riverside

The 3’ untranslated region (UTR) of mRNAs is extensively targeted by a myriad of RNA-binding proteins (RBPs) and microRNAs (miRNAs) for mRNA decay and translation control. HuR, a ubiquitously expressed RBP, shares many target mRNAs with miRNAs, and their combinatorial control of gene expression has been recently reported. However, the whole picture of this regulation paradigm across the transcriptome remains largely unclear. Here, we investigate the interactions between Ago2 (the core RBP in miRNA machinery) and HuR at the transcriptome-wide level. Using HITS-CLIP, we determined the binding locations and binding levels of HuR and Ago2 on mRNAs. To detect HuR-Ago2 interactions, we measured the CLIP signals of Ago2 while manipulating the protein level of HuR. Globally, occupancies of the most extreme changing Ago2 sites upon HuR knockdown were affected by the HuR’s presence on the same 3’UTRs, suggesting that HuR exerts influence on Ago2 binding to mRNAs. In addition, HuR and Ago2 sites largely overlap on 3’UTRs. To study HuR-miRNA interactions at individual sites, thirteen overlapping CLIP sites were selected, and their activities were tested by luciferase assays. Overall, we found three sites where HuR altered the miRNA repression effects, indicating their interaction at those sites. Additionally, we performed sequence analysis to determine the identities of three uncharacterized bands in HuR CLIP. Our results show that the composition of the sequences in these bands was different from the major band; instead, motif analysis implied that they might not be HuR bands, though higher depth sequencing data were required for confirmation. Taken together, we present an integrated approach to study the interactions between the miRNA machinery and HuR on a transcriptome-wide scale, and define the prevalence and versatility of their interactions on mRNA targets.