Diversity in mRNA closed-loop formation during translation

Abstract

A key feature of eukaryotic translation initiation is the 'closed-loop' model, which describes a process where the ends of an mRNA molecule, namely the 5' cap and the 3' poly(A) tail, are circularised by a bridge of protein factors - eIF4E, eIF4G and Pab1. Formation of this structure has long been proposed as an explanation for how mRNA end proximity might enhance translation. However, although initial studies concluded an important involvement of the closed-loop in productive translation, this has been challenged by more recent in vivo findings reporting low levels of closed-loop formation on some translating mRNAs and the potential for mRNA-dependent closed-loop formation.

To better understand characteristics of the mRNA closed-loop, we have developed an in vivo approach in Saccharomyces cerevisiae to probe the closed-loop transcriptome-wide. Combining formaldehyde crosslinking, immunoprecipitation, limited nuclease digestion and short-read RNA-sequencing, we captured the closed-loop and associated mRNAs. We then devised a bioinformatic method to define mRNA extremities and compare the relative association of mRNA ends to the closed-loop factors as a measure for an intact closed-loop protein bridge.

From our transcriptome-wide analyses, we find that the closed-loop likely can form in vivo. We are able to observe examples of mRNAs with significant enrichments at both ends upon immunoprecipitation with the 5' cap-associated closed-loop factor eIF4E. However, it is noteworthy that the closed-loop does not appear to be a consistently observed phenomenon at steady state. Instead, our research demonstrates that the extent of closed-loop formation varies greatly across the transcriptome. Some mRNAs exhibit a 'high closed-loop forming' pattern, characterised by nearly equal association of both ends with the closed-loop factors, while others demonstrate a 'low closed-loop forming' profile, with a more unequal distribution of their ends in relation to these factors.

Correlating the extent of closed-loop formation propensity to physical and functional mRNA features such as translation efficiency, we do not find a strong relationship between the various features tested and extent of closed-loop formation. Therefore, our findings prompt a more nuanced consideration of the role of the closed-loop in translation, and provide a methodology for further investigation of the closed-loop's biological functions. Show more