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

AGO2, is unique amongst the mammalian AGO proteins in that it has slicing activity (48) (49) (50) . In fact, AGO2 has been demonstrated to cleave a subset of pre-miRNAs, named AGO-cleaved pre-miRNAs (ac-pre-miRNAs) (13) . The best characterized of the ac-pre-miRNAs, pre-miR-451, is trimmed by PARN . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/294488 doi: bioRxiv preprint following AGO2 cleavage to generate the mature miRNA sequence (12, 14, 15, 51) . However, the extent to which this process occurs in mammalian pre-miRNA populations has not been determined. AGO2 cleavage events are known to occur around 10 nucleotide upstream of the 3' end of the pre-miRNA and give rise to 5p miRNAs (13) . To search for these events, we calculated the percentage of pre-miRNA-seq reads that terminated 8-15 nucleotide from the 3' end of the 3p miRNA for each miRNA. In mouse, for which ac-pre-miRNAs are best characterized, we observed all previously identified ac-pre-miRNAs with the exception of miR-451, which is not expressed in MEFs (Table 1 ). In our human dataset, we also observed previously identified ac-pre-miRNAs, including 9 members of the let-7 family and miR-9-2 (Table S3) . From this approach, we identified 7 putative ac-pre-miRNA candidates in humans and 37 in mouse, including hsa-miR-455 and mmu-miR-335 (Figs. 3A-B and Tables 1 and S3 ).