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

Nucleic Acids Research
Anastasiia KamenskaNancy Standart

Abstract

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. A...Continue Reading

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Methods Mentioned

BETA
transfection
PCR
two hybrid
immunoprecipitation
Assay
electrophoresis
restriction digests
transfections
protein assay
Fluorescence imaging

Software Mentioned

COFFEEE
DOMAC
Gene 6000
Emboss Needle
POODLE
PSIPRED
ClustalW
ImageQuant
EMBOSS
Jpred

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