Deep sequencing of microRNA precursors reveals extensive 3' end modification

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. An emerging mechanism to control miRNA production is the addition of an oligo-uridine tail to the 3' end of the precursor miRNA. This has been demonstrated for the Let-7 family of miRNAs in embryonic cells. Additionally, non-templated nucleotides have been found on mature miRNAs species, though in most cases it is not known if nucleotide addition occurs at the precursor step or at the mature miRNA. To examine the diversity of nucleotide addition we have developed a high throughput sequencing method specific for miRNA precursors. Here we report that non-templated addition is a widespread phenomenon occurring in many miRNA families. As previously reported, Let-7 family members are uridylated in embryonic cells; however, we find that the fraction of uridylated precursors increases with differentiation, and is highest in adult tissues, exceeding 30 percent of all sequence reads for some Let-7 family members. A similar fraction of sequence reads are modified for many other miRNA families. Mono-uridylation is most common, with cytidine and adenosine modification less frequent but occurring above the expected error rate for Illumina sequencing. Nucleotide addition in cell lines is associated with 3' end degradation, in contrast to adult tissues, where modification occurs predominantly on full-length precursors. This work provides an unprecedented view of the complexity of 3' modification and trimming of miRNA precursors.