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

Pre-miRNAs and miRNAs are post-transcriptionally modified at their 3' ends through the action of TUT4 and TUT7 terminal uriydyl transferases (TUTases) (17, 18, 46, 47) . We found that 14.5% of human and 17.4% of mouse pre-miRNAs contained single nucleotide additions to their 3' ends, whereas 4.9% and 7.1% had more than two non-templated additions on their 3' ends in human and mouse high confidence miRBase-annotated miRNAs, respectively (Figs. 2C and S2F). For mature miRNAs, we also found a large number of single nucleotide additions at the 3' end; 8.4% of human and 12.5% of mouse mature miRNAs. However, tails greater than one nucleotide were much less frequent, with only 1.1% of human and 2.2% of mouse mature miRNAs containing long tails. The lower overall modification level of mature miRNAs is consistent with the hypothesis that oligo-uridylation is a signal for degradation, whereas monouridylation establishes the 3' 2 nt overhang, required for efficient DICER cleavage.