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

High-throughput sequencing of total or AGO-bound smRNAs in numerous cells, tissues, and organisms has provided a wealth of information about miRNAs. In concert with bioinformatic approaches, these datasets have been expanded to identify thousands of novel miRNAs (25, 26) . In contrast, sequencing of pre-miRNAs has been challenging, due to the presence of other RNA species, including the highly abundant transfer RNAs (tRNAs) and small nucleolar RNAs (snoRNAs), that exist in the same size range as pre-miRNAs. Attempts to use size selection to sequence pre-miRNAs have achieved < 1% of total sequenced clones corresponding to pre-miRNAs even after selective depletion of abundant species (27) . Alternatively, primer-based approaches have been applied, but these methods restrict analysis to known miRNA species and thus cannot be . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 used for discovery (20, 21, 28) . Thus, there is a need for unbiased, sequencing based approaches to gain a more comprehensive understanding of pre-miRNA expression and sequence content.