Dynamic interaction of poly(A)-binding protein with the ribosome

Kodai Machida,Tomoaki Shigeta,Takuhiro Ito,Nahum Sonenberg,Yuki Yamamoto,Hiroaki Imataka,Yuri Svitkin

doi:10.1038/s41598-018-35753-1

Abstract

Eukaryotic mRNA has a cap structure and a poly(A) tail at the 5′ and 3′ ends, respectively. The cap structure is recognized by eIF (eukaryotic translation initiation factor) 4 F, while the poly(A) tail is bound by poly(A)-binding protein (PABP). PABP has four RNA recognition motifs (RRM1–4), and RRM1-2 binds both the poly(A) tail and eIF4G component of eIF4F, resulting in enhancement of translation. Here, we show that PABP interacts with the 40S and 60S ribosomal subunits dynamically via RRM2-3 or RRM3-4. Using a reconstituted protein expression system, we demonstrate that wild-type PABP activates translation in a dose-dependent manner, while a PABP mutant that binds poly(A) RNA and eIF4G, but not the ribosome, fails to do so. From these results, functional significance of the interaction of PABP with the ribosome is discussed.

Highlights

IntroductionMost eukaryotic mRNA has a cap structure with m7GpppN (where N is any nucleotide) at the 5′ end and a poly(A) tail structure at the 3′ end
Most eukaryotic mRNA has a cap structure with m7GpppN at the 5′ end and a poly(A) tail structure at the 3′ end
During purification of ribosomes from HeLa cells according to the procedure outlined in Fig. 1A, we found that poly(A)-binding protein (PABP) co-purified with ribosomes as described below

Summary

Introduction

Most eukaryotic mRNA has a cap structure with m7GpppN (where N is any nucleotide) at the 5′ end and a poly(A) tail structure at the 3′ end. The cap structure is bound by eukaryotic translation initiation factor (eIF) 4F, which comprises three proteins (eIF4E, eIF4A and eIF4G). EIF4E interacts directly with the cap, and eIF4A is an ATP-dependent RNA helicase thought to work with the RNA-binding initiation factor eIF4B to unwind the secondary structure of the 5′-untranslated region of the mRNA. EIF4G is a large modular scaffolding protein with binding sites for eIF4E, eIF4A and eIF3, a multi-subunit initiation factor that interacts directly with the small (40S) ribosomal subunit[1,2]. We report that PABP dynamically associates with the ribosomal RNAs, and this interaction might support translation

Methods

Results

Conclusion