pre-miRNA profiles obtained through application of locked nucleic acids and deep sequencing reveals complex 5'/3' arm variation including concomitant cleavage and polyuridylation patterns

Nucleic Acids Res. 2012 Feb;40(4):1424-37. doi: 10.1093/nar/gkr903. Epub 2011 Nov 3.

Abstract

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 3'- and 5'-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 pre-miRNA regulation and processing, and shed new light on overlooked products of miRNA processing pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • HeLa Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • MicroRNAs / chemistry*
  • MicroRNAs / metabolism
  • Nucleotide Motifs
  • Oligodeoxyribonucleotides / chemistry
  • Oligonucleotides / chemistry*
  • Poly U / analysis*
  • RNA Precursors / chemistry*
  • RNA Precursors / metabolism
  • RNA Processing, Post-Transcriptional*
  • RNA-Binding Proteins / metabolism
  • Sequence Analysis, RNA
  • Uridine / analysis
  • Uridine / metabolism

Substances

  • MicroRNAs
  • Oligodeoxyribonucleotides
  • Oligonucleotides
  • RNA Precursors
  • RNA-Binding Proteins
  • locked nucleic acid
  • Poly U
  • Uridine