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

For AGO-associated stem-loops derived from mouse mRNAs we mapped 2,698 miRNA-seq reads, however 71% of these mapped to a single locus in the CDS of Rpl9 (Figs. 6G-I) . Northern blotting with a probe complementary to this sequence revealed a strong signal at ~70 nt, corresponding to the stem-loop, and a weak signal at 25 nt, corresponding to the expected size of the novel miRNA. (Fig. 6J) . Therefore, we provide evidence that Rpl9 is processed into a stem-loop like structure and further processed, albeit with low efficiency, into a miRNA-like smRNA. Other AGO-bound smRNA producing stem-loops were regions of the Cyr61 5' UTR, Asf1b 5' UTR, and Klf9 3' UTR (Fig. S7) . Collectively, these data demonstrate that pre-miRNA-seq uncovers AGO-associated mRNA stem-loops, some of which may represent novel miRNAs, but most of which are poorly processed into AGO-bound smRNAs in the cell lines investigated in this study.