A key feature of eukaryotic translation initiation is the ‘closed-loop’ model, which describes a process by which the ends of an mRNA molecule, namely the 5’ cap and the 3’ poly(A) tail, are circularised by protein factors eIF4E, eIF4G and PABP in a manner leading to effective translation. Once thought to form in the majority of cases concerning translation of capped, polyadenylated mRNA, the ubiquity and necessity of the closed-loop complex during initiation has more recently been brought into question. One theory is that the closed-loop may represent an additional layer of translation initiation regulation by preferentially forming to varying extents on different mRNAs. As the closed-loop is implicated in a host of processes such as scanning, translational responses to stress and ribosome recycling, a differential association of mRNAs to the closed-loop factors would affect their respective translational outcomes.
To investigate whether closed-loop formation may be mRNA-dependent, we have developed an in vivo approach combining formaldehyde-crosslinking of S. cerevisiae followed by immunoprecipitation of the tagged closed-loop factors with their interacting mRNAs. Coupling this with RNA-Seq, we aim to use the relative association of mRNAs to the closed-loop factors as a proxy for their extent of closed-loop formation. By analysing closed-loop association across the transcriptome, we hope to elucidate whether closed-loop formation is influenced by mRNA-intrinsic features, and whether it represents an additional form of translation initiation control. In performing this study, we hope that better understanding of closed-loop complex formation during translation initiation will aid any future development of translation-targeting therapeutics.