Characterization of novel primary miRNA transcription units in human cells using Bru-seq nascent RNA sequencing

MicroRNAs (miRNAs) are key contributors to gene regulatory networks. Because miRNAs are processed from RNA polymerase II transcripts, insight into miRNA regulation requires a comprehensive understanding of the regulation of primary miRNA transcripts. We used Bru-seq nascent RNA sequencing and hidden Markov model segmentation to map primary miRNA transcription units (TUs) across 32 human cell lines, allowing us to describe TUs encompassing 1462 miRNAs from miRBase and 465 from MirGeneDB. We identified 54 TUs not previously described for MirGeneDB miRNAs. Many primary transcripts containing miRNA sequences failed to generate mature miRNAs, suggesting that miRNA biosynthesis is under both transcriptional and post-transcriptional control. In addition to constitutive and cell-type specific TU expression regulated by differential promoter usage, miRNA synthesis can be regulated by transcription past polyadenylation sites (transcriptional read through) and promoter divergent transcription (PROMPTs). We identified 203 miRNA TUs with novel promoters, 99 with transcriptional read-throughs and 3 miRNA TUs that resemble PROMPTs in at least one cell line. The miRNA TU annotation data resource described here reveals a greater complexity in miRNA regulation than previously known and provides a framework for identifying cell-type specific differences in miRNA transcription in cancer and following exposure to environmental insults or therapeutics. Nascent RNA Bru-seq used to map primary miRNA transcripts across 32 diverse human cell lines, allowing for systematic assessment of the various transcription units encompassing known miRNAs