Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory

Leveraging the knowledge that AGO is an integral component of the pre-miRNA processing complexes (miRLC and miPDC), we recently developed an approach to enrich for pre-miRNAs by immunoprecipitating AGO proteins and subsequently cloning RNAs from 50-80 nucleotides (nts). Using this approach, pre-miRNA libraries from mouse embryonic fibroblasts (MEFs) were generated with > 40% of reads mapping to miRNA loci (19) . Thus, this strategy can be used to efficiently study pre-miRNAs globally without the need for primer-based approaches or depletion of abundant RNA species. Here, we applied this approach to isolate and sequence pre-miRNAs and mature miRNAs from human embryonic kidney (HEK293T) cells. We developed a bioinformatic pipeline to capture post-transcriptional modifications, which we applied to data generated in this study from HEK293T cells, as well as to the previous data generated in MEFs. Our results provide global insights into pre-miRNA processing and provide an alternative strategy for identifying pre-miRNAs and other AGO-associated stem-loops in transcriptomes of interest.