Examples of novel mammalian 5′-tailed mirtrons annotated in this study. (A) Human NXF1 (hsa-mir-6514) contains a 5′-tailed mirtron, whose mature species were recovered in independent Ago-IP data sets. (B) Small RNAs mapped to murine Camk2a (mmu-mir-6982) define a 5′-tailed mirtron that, although modestly expressed, is very well-conserved among mammals. Curiously, the presumed seed sequences of both 5p and 3p species have diverged, but all of these changes are structurally conservative. Such evolutionary patterns suggest that the structure, rather than the precise sequence, of this mirtron hairpin is under selection. (C) A candidate 5′-tailed mirtron in the intron of human CUX2. While this hairpin generates a typical miRNA/star duplex with high 5′ specificity, the existence of only two star reads was below our requirement for confident annotation. (D) A locus that emerged from the control analysis of “shifted” intron coordinates. This hairpin spans an exon–intron junction of mmu-Pdgfra and generates a typical miRNA/star duplex, for which the dominant 3p species terminate in CAG or exhibit untemplated uridylation. This layout strongly suggests that this region of Pdgfra harbors a 5′-tailed mirtron (mmu-mir-7025) that bypasses an annotated 5′ splice acceptor and uses an unannotated 3′ splice acceptor; note that the CAG acceptor is conserved in many mammals. Overall, note that the 3p species of 5′-tailed mirtrons generally accumulate high levels of untemplated uridine (yellow highlight), with a lower level of untemplated adenine.
