Regulation of protein synthesis on mRNA is central to eukaryotic expression of genes. Regulatory inputs are specified by the mRNA untranslated regions (UTRs) and often target translation initiation. Initiation consists of dynamic and complex interactions between eukaryotic initiation factors (eIFs) and the small ribosomal subunit (SSU), and concludes with joining of the large ribosomal subunit (LSU) to form a full ribosome and translate the mRNA code. While control of protein synthesis is important for fast-paced cell adaptation, methods to study the mechanistic intricacies of translation initiation in vivo transcriptome-wide were lacking.
We developed translation complex profile sequencing (TCP-seq) [Archer SK et al. Nature 2016 535:570-574; Shirokikh NE et al. Nat. Protoc. 2017 12:697-731], a method related to the ribosome profiling approach. TCP-seq uniquely allows to resolve all translation intermediates, including the elusive mRNA ‘scanning’ by SSUs, visualize start codon recognition events, and capture diverse conformations of elongating ribosomes in vivo. Thus, the method can be used to pinpoint mRNAs with high potential of functional control and directly visualize locations of the regulatory elements in their 5'UTRs.
Combining TCP-seq with eIF-selective purification of complexes in yeast model system, we aim to discern the fundamental mechanistic problems of initiation, such as what proteins confer directionality to the SSUs during scanning, and how ribosomal recruitment to mRNA is controlled during nutrient stress conditions. We further adapt TCP-seq to use in mammalian cells and aim to obtain snapshots of translation in normal and cancer cells to identify 5'UTRs which can be selectively up- or downregulated in malignancies.