Functional Cyclization of Eukaryotic mRNAs.

Olga Alekhina,Sergey Dmitriev,Ilya Terenin,Konstantin Vassilenko

doi:10.3390/ijms21051677

Abstract

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

Highlights

More than 50 years ago, electron micrograph images revealed a complex topology of translating polyribosomes
The time lag between the start of the reaction and the appearance of a full-length, active luciferase can be used to estimate the time required for the synthesis of a complete protein molecule during a full round of translation consisting of initiation, elongation, and termination
The latter explanation is supported by well documented evidence of functional mRNA cyclization in the absence of the canonical cap/eIF4E/eIF4G/poly(A)-binding protein (PABP)/poly(A) bridge, e.g., in the cases of non-polyadenylated rotaviral and histone mRNAs, or internal ribosome entry site (IRES)-containing picornavirus and flavivirus genomic mRNAs

Summary

Introduction

More than 50 years ago, electron micrograph images revealed a complex topology of translating polyribosomes. The so-called closed-loop model for mRNA circularization by means of bridging 5’ and 3 ́ termini was proposed [6], well before the discovery of the underlying protein interactions [7,8]. The direct interaction between the mRNA termini was proved by the discovery of the physical association between the initiation factor eIF4G and the poly(A)-binding protein (PABP) in yeast [12,13], plants [14], and mammals [15]. Atomic force microscopy experiments demonstrated the formation of RNA circles when a model capped and polyadenilated RNA was mixed with yeast eIF4F (eIF4E+eIF4G) and PABP, whereas the absence of any of these proteins or their inability to bind eIF4E or PABP prevented RNA looping [13]. It is possible that other protein factors or ribosomes themselves are involved in the formation of an ordered polysome structure bringing mRNA 5 and 3 termini together [17,18]

Methods

Results

Conclusion