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A chemiluminescence-based reporter system to monitor nonsense-mediated mRNA decay.

Abstract：

:Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that mediates rapid degradation of transcripts bearing premature translation termination codons (PTCs) and thereby limits the expression of unproductively processed mRNAs and the synthesis of C-terminally truncated peptides. Both its importance as a means to control gene expression and in the context of genetic and acquired human diseases call for an exploration of the mammalian NMD pathway using chemical biology approaches. Here, we describe a novel cell-based chemiluminescence reporter system that recapitulates the hallmark features of mammalian NMD. The assay is characterized by its high sensitivity, robustness, and its potential for automated handling. Limiting NMD efficiency by RNAi-mediated depletion of the essential NMD factor UPF1 markedly and specifically increased the NMD reporter mRNA level and resulted in a proportional increase in protein expression reflected by Renilla luminescence. The PI 3-kinase inhibitor wortmannin has previously been found to up-modulate PTC-containing transcripts by inhibiting the UPF1 kinase SMG1. Wortmannin treatment enhanced NMD reporter expression in our system in a dose-dependent way, illustrating its utility for small molecule screening.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Boelz S,Neu-Yilik G,Gehring NH,Hentze MW,Kulozik AE

doi

10.1016/j.bbrc.2006.08.017

subject

Has Abstract

pub_date

2006-10-13 00:00:00

pages

186-91

issue

1

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(06)01812-2

journal_volume

349

pub_type

杂志文章

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