MicroRNAs Control Translation Initiation by Inhibiting Eukaryotic Initiation Factor 4E/Cap and Poly(A) Tail Function

MicroRNAs (miRNAs) repress translation of target mRNAs by interaction with partially mismatched sequences in their 3' UTR. The mechanism by which they act on translation has remained largely obscure. We examined the translation of mRNAs containing four partially mismatched miRNA-binding sites i...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 47; pp. 16961 - 16966
Main Authors Humphreys, David T, Westman, Belinda J, Martin, David I K, Preiss, Thomas
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 22.11.2005
National Acad Sciences
Subjects
Binding sites
Biochemistry
Biological Sciences
Cricket paralysis virus
Encephalomyocarditis virus
Eukaryotic Initiation Factor-4E - antagonists & inhibitors
Eukaryotic initiation factors
HeLa Cells
Humans
Messenger RNA
MicroRNA
MicroRNAs - physiology
Poly(A)-Binding Proteins - antagonists & inhibitors
Proteins
Receptors, CXCR4 - genetics
Repression
Ribonucleic acid
Ribosomes - physiology
RNA
RNA Caps - antagonists & inhibitors
RNA stability
RNA, Messenger - metabolism
Standard deviation
Transfection
Untranslated regions
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Summary:MicroRNAs (miRNAs) repress translation of target mRNAs by interaction with partially mismatched sequences in their 3' UTR. The mechanism by which they act on translation has remained largely obscure. We examined the translation of mRNAs containing four partially mismatched miRNA-binding sites in the 3' UTR in HeLa cells cotransfected with a cognate miRNA. The mRNAs were prepared by in vitro transcription and were engineered to employ different modes of translation initiation. We find that the 5' cap structure and the 3' poly(A) tail are each necessary but not sufficient for full miRNA-mediated repression of mRNA translation. Replacing the cap structure with an internal ribosome entry site from either the cricket paralysis virus or the encephalomyocarditis virus impairs miRNA-mediated repression. Collectively, these results demonstrate that miRNAs interfere with the initiation step of translation and implicate the cap-binding protein eukaryotic initiation factor 4E as a molecular target.
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Conflict of interest statement: No conflicts declared.
To whom correspondence should be addressed. E-mail: t.preiss@victorchang.unsw.edu.au.
This paper was submitted directly (Track II) to the PNAS office.
Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA
Author contributions: D.T.H. and B.J.W. performed research; D.T.H. and B.J.W. analyzed data; D.I.K.M. and T.P. designed research; and D.I.K.M. and T.P. wrote the paper.
Abbreviations: CrPV, cricket paralysis virus; eIF, eukaryotic initiation factor; EMCV, encephalomyocarditis virus; F-luc, firefly luciferase; IRES, internal ribosomal entry site; miRNA, microRNA; P-bodies, processing bodies; PABP, poly(A)-binding protein; R-luc, Renilla luciferase.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0506482102