Translation complex stabilization on messenger RNA and footprint profiling to study the RNA responses and dynamics of protein biosynthesis in the cells.

During protein biosynthesis, ribosomes bind to messenger (m)RNA, locate its protein-coding information, and translate the nucleotide triplets sequentially as codons into the corresponding sequence of amino acids, forming proteins. Non-coding mRNA features, such as 5' and 3' untranslated regions (UTRs), start sites or stop codons of different efficiency, stretches of slower or faster code and nascent polypeptide interactions can alter the translation rates transcript-wise. Most of the homeostatic and signal response pathways of the cells converge on individual mRNA control, as well as alter the global translation output. Among the multitude of approaches to study translational control, one of the most powerful is to infer the locations of translational complexes on mRNA based on the mRNA fragments protected by these complexes from endonucleolytic hydrolysis, or footprints. Translation complex profiling by high-throughput sequencing of the footprints allows to quantify the transcript-wise, as well as global, alterations of translation, and uncover the underlying control mechanisms by attributing footprint locations and sizes to different configurations of the translational complexes. The accuracy of all footprint profiling approaches critically depends on the fidelity of footprint generation and many methods have emerged to preserve certain or multiple configurations of the translational complexes, often in challenging biological material. In this review, a systematic summary of approaches to stabilize translational complexes on mRNA for footprinting is presented and major findings are discussed. Future directions of translation footprint profiling are outlined, focusing on the fidelity and accuracy of inference of the native in vivo translation complex distribution on mRNA.