Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5'-seed/ cleavage/anchor regions and stabilize predicted mmu-let-7a:mRNA duplexes

Genome Res. 2008 Oct;18(10):1571-81. doi: 10.1101/gr.078246.108. Epub 2008 Jul 9.

Abstract

Massively parallel sequencing of millions of < 30-nt RNAs expressed in mouse ovary, embryonic pancreas (E14.5), and insulin-secreting beta-cells (betaTC-3) reveals that approximately 50% of the mature miRNAs representing mostly the mmu-let-7 family display internal insertion/deletions and substitutions when compared to precursor miRNA and the mouse genome reference sequences. Approximately, 12%-20% of species associated with mmu-let-7 populations exhibit sequence discrepancies that are dramatically reduced in nucleotides 3-7 (5'-seed) and 10-15 (cleavage and anchor sites). This observation is inconsistent with sequencing error and leads us to propose that the changes arise predominantly from post-transcriptional RNA-editing activity operating on miRNA:target mRNA complexes. Internal nucleotide modifications are most enriched at the ninth nucleotide position. A common ninth base edit of U-to-G results in a significant increase in stability of down-regulated let-7a targets in inhibin-deficient mice (Inha-/-). An excess of U-insertions (14.8%) over U-deletions (1.5%) and the presence of cleaved intermediates suggest that a mammalian TUTase (terminal uridylyl transferase) mediated dUTP-dependent U-insertion/U-deletion cycle may be a possible mechanism. We speculate that mRNA target site-directed editing of mmu-let-7a duplex-bulges stabilizes "loose" miRNA:mRNA target associations and functions to expand the target repertoire and/or enhance mRNA decay over translational repression. Our results also demonstrate that the systematic study of sequence variation within specific RNA classes in a given cell type from millions of sequences generated by next-generation sequencing (NGS) technologies ("intranomics") can be used broadly to infer functional constraints on specific parts of completely uncharacterized RNAs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cells, Cultured
  • Deoxyuracil Nucleotides / metabolism
  • Embryo, Mammalian / metabolism
  • Female
  • Mice
  • MicroRNAs / chemistry*
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • RNA Editing*
  • RNA Nucleotidyltransferases / metabolism
  • RNA Stability
  • RNA, Guide / metabolism
  • RNA, Messenger / metabolism*

Substances

  • Deoxyuracil Nucleotides
  • MicroRNAs
  • RNA, Guide
  • RNA, Messenger
  • deoxyuridine triphosphate
  • RNA Nucleotidyltransferases
  • UTP-RNA uridylyltransferase