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Nucl. Acids Res.-2014-Kamenska-3298-313.pdf (4.3 MB)

Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing.

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posted on 2015-04-27, 14:38 authored by A. Kamenska, Wei-Ting Lu, D. Kubacka, H. Broomhead, N. Minshall, Martin Bushell, N. Standart
A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

Funding

Wellcome Trust [084885/Z/08/Z] and BBSRC [BB/ J00779X/1 to N.S.]; MRC [G0902052 to M.B.]; Gates Cambridge Scholarship postgraduate funding (to A.K.). Funding for open access charge: Wellcome Trust and BBSRC.

History

Citation

Nucleic Acids Research, 2014, 42 (5), pp. 3298-3313

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/School of Biological Sciences/Department of Biochemistry

Version

  • VoR (Version of Record)

Published in

Nucleic Acids Research

Publisher

Oxford University Press (OUP)

issn

0305-1048

eissn

1362-4962

Copyright date

2013

Available date

2015-04-27

Publisher version

http://nar.oxfordjournals.org/content/42/5/3298

Notes

PMCID: PMC3950672 Supplementary Data are available at NAR Online, including [44].

Language

en

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