The Interaction between Cap-binding Complex and RNA Export Factor Is Required for Intronless mRNA Export

Masatoshi Hagiwara,Hiroshi Kimura,Tetsuro Hirose,Takayuki Nojima

doi:10.1074/jbc.m700629200

Abstract

RNA export factor (REF) is a component of the exon junction complex (EJC) that is deposited on mRNA in a splicing-dependent manner, and targets spliced mRNA for export. In this study, analysis of the RNA-binding protein complexes revealed that REF associates with beta-globin mRNA at the region other than the EJC deposition site. Comparison between RNA polymerase II and T7 transcription and further analysis showed that the deposition of REF apart from the EJC is dependent on the 5' cap structure, but not splicing. Excess amounts of m(7)GpppG cap analog reduced REF binding to intronless mRNA, and a co-immunoprecipitation experiment revealed that REF interacts with the cap-binding protein CBP20. The export of Cy3-labeled intronless beta-globin mRNA from nuclei of HeLa cells was enhanced by co-injection of CBP20 and REF. Thus, REF recruited by CBP20 may play a stimulatory role to export the capped intronless mRNAs.

Highlights

The transport of mRNA from the nucleus to the cytoplasm is linked to pre-mRNA splicing, especially in metazoans [6]
RNA export factor (REF) Binds to mRNA Independently of the exon junction complex (EJC) Deposition— To analyze transitions in mRNA-protein complex (mRNP) composition during mRNA biogenesis in the nucleus, we developed a coupled in vitro system for examining transcription-splicing reactions
To determine whether REF is recruited as an EJC component, we examined the EJC deposition on spliced mRNA originating from T7 and RNA polymerase II (RNAPII) transcripts

Summary

Introduction

The transport of mRNA from the nucleus to the cytoplasm is linked to pre-mRNA splicing, especially in metazoans [6]. As the EJC proteins (REF, Y14, SRm160, UAP56, RNPS1, and Magoh) are accumulated at sites of transcription [31], we immunoprecipitated in vitro transcribed and spliced ␤-globin mRNA using antibodies against the EJC components and examined whether RNAPII transcription enhances the EJC deposition or not.

Results

Conclusion