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

  1. Jeffrey G. Reid1,
  2. Francis C. Lynn2,
  3. Ankur K. Nagaraja1,
  4. Rafal B. Drabek1,
  5. Donna M. Muzny1,
  6. Chad A. Shaw1,
  7. Michelle K. Weiss3,
  8. Arash O. Naghavi3,
  9. Mahjabeen F. Khan3,
  10. Huifeng Zhu3,
  11. Gemunu H. Gunaratne3,
  12. David B. Corry1,
  13. Jonathan N. Miller1,
  14. Michael S. German2,
  15. Richard A. Gibbs1,
  16. Martin M. Matzuk1, and
  17. Preethi H Gunaratne3,4
  1. 1 Baylor College of Medicine;
  2. 2 UCSF Diabetes Center;
  3. 3 University of Houston

Abstract

Massively parallel sequencing of millions of <30 nt RNAs expressed in mouse ovary, embryonic pancreas (E14.5) and insulin-secreting beta-cells (βTC-3) reveals that ~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 exhibits 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 9th nucleotide position. A common 9th base edit of U-to-G results in a significant increase in stability of down regulated let-7a targets in inhibin knockout mutants (Inha-/-). An excess of U-insertions (14.8%) over U-deletions (1.5%) and the presence of cleaved intermediates suggests that a mammalian TUTase (terminal uridylyl transferase) mediated dUTP-dependent U-insertion/U-deletion cycle maybe 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.

Footnotes

    • Received March 11, 2008.
    • Accepted June 27, 2008.

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  1. Published in Advance July 9, 2008, doi: 10.1101/gr.078246.108 Genome Res. gr.078246.108 Copyright © 2008, Cold Spring Harbor Laboratory Press

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