From synaptic plasticity including memory functions, to adaptation to immediate environments and stresses, eukaryotic cells rely on fast-paced translational control. Cancer cells also exhibit distinct translational profiles. Many protein factors involved in translation are known oncogenes, or their functions are dysregulated in malignancies. Thus, to understand how the cells mitigate adverse conditions or acquire and maintain a malignant phenotype, it is critical to expose the underlying regulation of protein synthesis from mRNA. To detect and investigate translational responses in vivo, 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 those of the elusive mRNA ‘scanning’ process, and visualize start codon recognition events [Shirokikh NE and Preiss T 2018 WIREs RNA 9: e1473], as well as capture diverse conformations of elongating and terminating ribosomes in vivo.
To address fast stress responses, we apply TCP-seq to investigate the dynamics of budding yeast translation upon removal of glucose. Yeasts depend on glucose as primary nutrient and acutely respond to change of its levels. We combine TCP-seq with translation initiation factor-selective purification of complexes to discern how glucose starvation response is initiated, maintained and released upon glucose re-supply in yeast. To inspect dysregulation of translation in cancer, we aim to investigate translation complex profiles of Eµ-myc mouse spontaneous lymphoma cells. We also use treatment of HEK 293 cells with inhibitors of translation initiation/prospective anti-cancer drugs, to model translational responses when different initiation stages are affected and identify mRNAs and mechanisms that escape these regulations. Along the way, we improve and simplify the TCP-seq technique.