Recent research hints at an underappreciated complexity in pre-miRNA processing and regulation.Global profiling of pre-miRNA and its potential to increase understanding of the pre-miRNA landscape is impeded by overlap with highly expressed classes of other non coding (nc) RNA. Here, we present a data set excluding these RNA before sequencing through locked nucleic acids (LNA), greatly increasing pre-miRNA sequence counts with no discernable effect on pre-miRNA or mature miRNA sequencing. Analysis of profiles generated in total, nuclear and cytoplasmic cell fractions reveals that pre-miRNAs are subject to a wide range of regulatory processes involving loci-specific 30- and 50-end variation entailing complex cleavage patterns with co-occurring polyuridylation. Additionally, examination of nuclear-enriched flanking sequences of pre-miRNA, particularly those derived from polycistronic miRNA transcripts, provides insight into miRNA and miRNA-offset (moRNA) production, specifically identifying novel classes of RNA potentially functioning as moRNA precursors. Our findings point to particularly intricate regulation of the let-7 family in many ways reminiscent of DICER1-independent, pre-mir-451-like processing, introduce novel and unify known forms of premiRNA regulation and processing, and shed new light on overlooked products of miRNA processing pathways.
