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.
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.
Reid
Jeffrey G
JG
Department of Chemistry, University of Houston, Houston, Texas 77204, USA.
Nagaraja
Ankur K
AK
Lynn
Francis C
FC
Drabek
Rafal B
RB
Muzny
Donna M
DM
Shaw
Chad A
CA
Weiss
Michelle K
MK
Naghavi
Arash O
AO
Khan
Mahjabeen
M
Zhu
Huifeng
H
Tennakoon
Jayantha
J
Gunaratne
Gemunu H
GH
Corry
David B
DB
Miller
Jonathan
J
McManus
Michael T
MT
German
Michael S
MS
Gibbs
Richard A
RA
Matzuk
Martin M
MM
Gunaratne
Preethi H
PH
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D058727:RNA, Small Untranslated
D003856:Deoxyuracil Nucleotides; D035683:MicroRNAs; D012333:RNA, Messenger; C027078:deoxyuridine triphosphate; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D058727:RNA, Small Untranslated