Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study

Zhanna A Afonina,Alexander G Myasnikov,Alexander S Spirin,Vladimir A Shirokov,Bruno P Klaholz

doi:10.1093/nar/gku599

Abstract

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3′-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5′- and 3′-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.

Highlights

Polyribosomes were discovered at the beginning of the 1960s as the main form of organization of translating ribosomes [1,2,3,4,5,6]
The observations of circular forms of eukaryotic polyribosomes were consistent with the experiments providing evidence for circular translation of messenger ribonucleic acid (mRNA) when ribosomes moving along the mRNA chain were not released after termination, supposedly reinitiating a new round of translation on the same mRNA chain [13,14,15]
Using the technique of cryo electron tomography, we demonstrate the formation of circular polyribosomes on this uncapped mRNA without poly(A) tail in a wheat germ cell-free translation system

Summary

Introduction

Polyribosomes were discovered at the beginning of the 1960s as the main form of organization of translating ribosomes [1,2,3,4,5,6]. A number of early electron microscopy observations demonstrated the circular array of ribosomes in eukaryotic polyribosomes [8,9,10] [see later publication, [11,12], for membrane-bound circular polyribosomes]. The capability of the cap-binding initiation factor eIF4F located at the capped 5 -end of mRNA to physically interact with the 3 located PABP and the resultant circularization of isolated mRNA chain under in vitro conditions were experimentally demonstrated [23]. Those results were in agreement with the ‘closed-loop model’ that implied the initiation-coupled circularization of capped polyadenylated mRNAs [(24); see [25]]. Since the model of cyclic polyribosome formation on the eukaryotic mRNAs by circularization due to joining of their 5 and 3 regions via the protein-toprotein (eIF4F-PABP) bridges was widely recognized and

Methods

Results

Conclusion