Integrating tRNA gene epigenomics and expression with codon usage unravels an intricate connection with translatome dynamics in .

Unlabelled: Codon usage bias impacts protein expression across all kingdoms of life, including trypanosomatids. These protozoa, such as the , primarily regulate their protein-coding genes through posttranscriptional mechanisms. Here, we integrated codon usage analyses with translatome data to investigate whether codon bias affects translated transcript expression levels in life forms. For the first time in trypanosomatids, tRNA sequencing was employed to reveal coadaptation between codon usage and anticodon availability. Despite notable differences in the proteomes of infective and noninfective forms, they exhibited similar pools of tRNAs and similar codon usage preferences, with notable differences in A-site ribosome occupancies. We developed pipelines to measure the adaptation of codons to their corresponding tRNA abundance pool (GM-tECA-Geometric Mean of tRNA Expression-Codon Adaptation) and to calculate the percentage of anticodon:codon base pairing modes in the genome. Our pipelines revealed an association between tRNA abundance, anticodon:codon pairing modes, and translated transcript levels. Highly expressed mRNAs are more adapted to tRNA abundance and favor either Watson-Crick or inosine pairing, whereas less expressed mRNAs exhibit lower adaptation to tRNA abundance and enrichment of codon with Wobble (G:U) pairing. Additionally, we observed that open chromatin levels of tRNA genes correlate with tRNA expression in noninfective forms, but not in infective forms, suggesting chromatin states do not control the tRNA pool in the latter. Overall, our findings suggest that protein expression in life forms is influenced by a combination of codon usage bias, tRNA abundance, and anticodon:codon pairing modes, but differences in ribosome A-site occupancies between life forms likely reflect additional layers of translation regulation.

Importance: Trypanosomatids primarily regulate protein expression at the posttranscriptional level, with codon bias playing a crucial role in controlling protein production across all life forms. This study investigated how codon usage, tRNA abundance, and codon pairing modes influence protein production in . Through tRNA sequencing and the integration of epigenomic and translatome data, we discovered that infective and noninfective forms of exhibit similar codon usage and tRNA pool preferences, despite having different proteomes. We developed pipelines applicable to any organism to measure codon adaptation to tRNA pools and pairing modes. Our analysis revealed that highly expressed genes are better aligned with more abundant tRNAs and favor Watson-Crick or inosine pairing. These findings suggest an additional layer of gene regulation based on tRNA availability and pairing modes, which impacts protein expression in the different life forms of .