The development of ALICE-tRNA-sequencing and its use in exploring the role of tRNAs in translational control


Sustaining proliferative signalling and loss of translational control is arguably the most fundamental trait of cancer cells, enabling tumour growth and metastatic dissemination. Transfer RNAs (tRNAs) have long been considered abundant “housekeeping” RNAs, functioning to decipher the universal genetic code. However, exhaustive analyses have implicated tRNA participation in a host of regulatory networks including the cellular stress response and protein synthesis. Recent findings suggest that the expression of tRNAs for synonymous codon usage is dependent on the differentiation/proliferation status of the cell and are coordinated with changes in translation. Although the molecular mechanisms that govern these changes are yet to be elucidated, cellular tRNA composition potentially introduces an additional layer of translational control. tRNAs are the most post-transcriptionally modified RNA species, with well over 50 unique modifications identified in eukaryotes. Consequently, isoacceptor identification and the measuring of the tRNA pool using next generation sequencing has long been an area of interest, with many attempts being made in literature. Using the Escherichia coli dealkylating enzyme AlkB and the novel tRNA high throughput sequencing methodology ALICE-tRNA-seq, we have developed a methodology that can accurately measure relative tRNA pools in vitro and in vivo. We show how other published tRNA sequencing protocols show bias towards tRNA sub populations, with our method showing a more realistic distribution across all tRNAs. We also show relative distribution changes in cellular and genetically modified mouse models of cancer, opening up a high resolution approach to establish the role of tRNAs in translational control and cell fate decisions

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