Integrating Ribosome Profiling and tRNA Sequencing with Computational Solutions to Decode the Complexity of the Translatome

Translation regulation is fundamental to cellular protein synthesis and plays critical roles in development, stress responses, and disease pathogenesis. While ribosome profiling has revolutionized our understanding of translation, current computational tools remain fragmented, requiring multiple disconnected pipelines for comprehensive analysis. Moreover, studying transfer RNA dynamics, particularly from translationally active populations, remains technically challenging due to labor-intensive isolation procedures and limitations in simultaneously capturing tRNA abundance, modifications, and fragmentation patterns. This thesis addresses these challenges through the development of two complementary tools: Martian, an integrated ribosome profiling analysis pipeline, and tRIBO-seq, a novel method for profiling ribosome-associated tRNAs. Martian is a comprehensive computational pipeline that consolidates five essential modules into a unified workflow: database creation with reference filtering and UTR correction, ribosome profiling with P-site mapping and coverage analysis, condition comparison with differential translation analysis, translation efficiency calculation integrating RNA-seq and Ribo-seq data, and nano-tRNAseq integration for tRNA quantification. Bench- marking against established tools (riboWaltz and Plastid) across 11 samples demonstrated that Martian achieves the fastest runtime (1,896 seconds versus 3,029 and 2,128 seconds), competitive P-site detection accuracy (TIS score 0.91 versus 1.00 and 0.14), and intermediate memory requirements. The pipeline successfully integrates multiple omics data types within a reproducible framework, providing researchers with an efficient platform for comprehensive translational analysis. tRIBO-seq (transfer RNA Ribosome Profiling by nanopore sequencing) introduces a streamlined method for profiling translationally active tRNAs by combining RiboLace-based ribosome isolation with nanopore direct RNA sequencing. This approach eliminates labor-intensive sucrose gradient fractionation, requires minimal input material (3-5 µg RNA from approximately 5 million cells), and can be completed within 5 hours. Critically, tRIBO-seq enables simultaneous quantification of tRNA abundance, post-transcriptional modifications, and fragmentation patterns from a single experiment. Application of tRIBO-seq to diverse stress conditions revealed distinct tRNAome reprogramming patterns. Under steady-state conditions, ribosome-associated and total tRNA pools showed high similarity with specific enrichment of initiator methionine tRNA in ribosomes. Amino acid deprivation (leucine or arginine) induced changes primarily in tRNA abundances with enrichment of cognate tRNA isoacceptors. Methionine starvation, in contrast, caused widespread tRNA hypomethylation linked to S-adenosylmethionine depletion, with minimal abundance changes. Oxidative stress induced by arsenite exposure triggered selective tRNA fragmentation predominantly in ribosome-embedded tRNAs, with cleavage occurring at anticodon regions, consistent with ribosome-localized angiogenin activation. These findings demonstrate that ribosome-associated tRNAs respond distinctly from total tRNA pools under stress conditions, emphasizing the importance of studying translationally active tRNA populations. Together, Martian and tRIBO-seq provide the research community with accessible, robust platforms for investigating translation regulation at both ribosome and tRNA levels, enabling comprehensive analysis of protein synthesis dynamics in health and disease.