Inhibition of Nonsense-mediated mRNA Decay by the Natural Product Pateamine A through Eukaryotic Initiation Factor 4AIII

Yongjun Dang,Woon-Kai Low,Jing Xu,Niels H Gehring,Harry C Dietz,Daniel Romo,Jun O Liu

doi:10.1074/jbc.m109.009985

Abstract

Nonsense-mediated mRNA decay (NMD) in mammalian cells is a key mechanism for the removal of mRNA containing premature stop codons and is mediated by the coordinated function of numerous proteins that dynamically associate with the exon junction complex. The information communicated by these interactions and the functional consequences from a mechanistic perspective, however, are not completely documented. Herein, we report that the natural product pateamine A (PatA) is capable of inhibiting NMD through direct interaction with eIF4AIII, which is independent of its inhibition of translation initiation. Furthermore, we have characterized the mechanisms by which PatA and cycloheximide modulate NMD. Unlike CHX, PatA was found to inhibit NMD by a novel mechanism that is independent of the phosphorylation of Up-frameshift protein 1.

Highlights

The proposition of the “linear interaction model” [4, 7]
To examine if Pateamine A (PatA) affected Nonsense-mediated mRNA decay (NMD), we employed the TCR-␤ minigene model system [17, 39]. These reporter genes were stably integrated into HeLa cells expressing transcripts without (TCRWT) or with a premature stop codons (PTCs) (TCRPTC) (Fig. 1A), and mRNA level was monitored by Northern blot analysis after drug treatment
We have demonstrated that PatA is capable of inhibiting NMD of mRNA through directly targeting eIF4AIII, a core component of the exon junction complex (EJC)

Summary

Introduction

The proposition of the “linear interaction model” [4, 7]. According to this model, deposition of the EJC onto mRNA causes the Y14-Magoh and eIF4AIII complex to effectively recruit Upf that interacts with Upf. Some studies have suggested that ribosome scanning that displaces EJCs occurs during the “pioneer round” of translation mediated by the association of nuclear cap-binding proteins (CBPs) such as CBP20 and CBP80 with NMD-targeted mRNAs as opposed to the subsequent “steady-state” translation via eukaryotic translation initiation factor 4A [21]. We found that PatA affected the dynamic association of Upf to the EJC without affecting Upf phosphorylation This is in contrast to cycloheximide (CHX), a translation elongation inhibitor that inhibits NMD. Together, these results shed new light on the regulation of NMD by the EJC and Upf proteins and suggest that PatA may serve as a new molecular probe of the NMD process

Methods

Results

Conclusion