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

We next calculated the miRNA-seq and smRNA-seq coverage at AGOassociated stem-loops derived from mRNAs. We found that miRNA-seq and smRNA-seq reads mapping to AGO-associated stem-loops derived from mRNAs were of a similar size as miRBase miRNAs and had non-templated additions to their 3' ends (Figs. 6A-B and S6A-D) . However, when we calculated the MPAR scores for AGO-associated stem-loops derived from mRNAs, we found that they had overall very low scores, indicating that they are poor substrates for producing mature AGO-bound smRNAs (Figs. 6C and S6E-F and Table S8 ). This is in agreement with the finding that these elements are more commonly oligo-tailed compared to canonical pre-miRNAs. In fact, we only found a total of 171 miRNAseq reads mapping to AGO-associated stem-loops derived from human mRNAs and the majority of these reads mapped to the recently identified miRNAs embedded within the mRNAs; GNAS, GLUL, and E2F1 (26,55) (example of . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 E2F1 in Figs. 6D-E) . Additionally, we found a total of 2,240 smRNA-seq reads mapping to AGO-associated mRNA stem-loops derived from human mRNAs, which in part reflects the higher sequencing depth of these libraries. Some of stem-loops with the most smRNA-seq reads, corresponded to previously identified novel miRNAs, including BRD2, GLUL and E2F1 (example in Fig. 6F) .