A key feature of eukaryotic translation initiation is the ‘closed-loop’ model, which describes a process whereby 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 PABP. Formation of this structure is thought to bring about more effective translation by stabilising the initiation complex and allowing efficient ribosome recycling. However, though once thought to form in most translation events concerning capped, polyadenylated mRNA, the ubiquity of the closed-loop complex during translation has been brought into question by studies showing low levels of closed-loop formation on certain translating mRNAs.
To investigate whether closed-loop formation may be mRNA-dependent, we have developed an in vivo approach in Saccharomyces cerevisiae. Combining formaldehyde crosslinking, immunoprecipitation and short-read RNA-sequencing, we compared the relative association of each mRNA’s 5’ and 3’ ends to the closed-loop factors. Equal associations of the two mRNA ends to the closed-loop proteins suggest frequent formation of an intact loop, while unequal associations of the ends suggest lower extents of loop formation. Overall, by using this metric, our research demonstrates that the extent of closed-loop formation varies greatly across the transcriptome. mRNAs ranged from being ‘high closed-loop forming’ with near-equal association of their two ends to the closed-loop factors, to ‘low closed-loop forming’ with more unequal association of their ends to the factors.
Further work is underway to determine whether the extent of closed-loop formation is correlated with specific mRNA-intrinsic features, functions or translation efficiency. If such links are established, this may indicate the possibility of closed-loop formation as a mechanism to control the efficiency of protein synthesis from different mRNAs. Determining a link between closed-loop formation, mRNA-intrinsic features and translation efficiency will be fundamental to development of more efficient mRNA therapeutics in the future.