<b>Hidden Diversity in Yeast tRNAs: Comparative Genomics and Modification Mapping in a Eukaryotic Subphylum</b>

Abstract: tRNA are adapter molecules with an integral role in translation and further roles in stress adaptation. Processing of tRNA is tightly regulated and includes the enzymatic addition of several post-transcriptional modifications that are required for translation efficiency, recognition, selective translation, and structure. We currently lack a multi-species wide view of tRNA modifying enzymes across eukaryotes. Here, we performed a comparative analysis of tRNA gene sequence, modification enzymes, and modification profiles across the Saccharomycotina subphylum. We employed machine learning methods to explore tRNA sequence conservation and to annotate modifying enzymes known to exist in fungi, humans, and prokaryotes. We then applied Nano-tRNAseq to three species (Saccharomyces cerevisiae, Hanseniaspora uvarum, and Yarrowia lipolytica) to profile modification signatures and compare modification patterns. We identified substantial lineage-specific conservation of tRNA sequences despite the highly conserved tRNA structure. We found significant variation in tRNA modifying enzyme repertoires across Saccharomycotina, including lineage-specific losses, and annotated a prokaryotic-associated enzyme, tilS. Integrating genomic and sequencing data enabled us to link enzyme repertoires with tRNA gene sequences. tRNA sequencing revealed distinct modification signatures across the three focal species, and further analysis using General Linearized modelling suggested tRNA enzyme loss is associated with target tRNA nucleotide absence in gene sequences. This work provides the first integrated view of tRNA gene and modification diversity in eukaryotes and expands the field of tRNA diversity in fungi. Supplementary Data index Supplementary Data 1. tRNA gene sequences for all 1154 Saccharomycotina species. Supplementary Data 2. Additional Random Forest model data for classification of any given tRNA from an isotype group into phylogenetic clade. Supplementary Data 3. Aligned tRNA gene sequences in Sprinzl position format. Supplementary Data 4. HMM annotation results for known fungal tRNA modifications in all 1154 Saccharomycotina species. Supplementary Data 5. HMM annotation results for galQ and manQ tRNA modifications in all 1154 Saccharomycotina species. Supplementary Data 6. HMM annotation results for known human tRNA modifications in all 1154 Saccharomycotina species. Supplementary Data 7. HMM annotation results for known prokaryotic tRNA modifications in all 1154 Saccharomycotina species. Supplementary Data 8. Raw Nanopore sequencing files in fastq format. Supplementary Data 9. Basecall error results for all tRNA in S. cereivisiae, H. uvarum and Y. lipolytica grouped by anticodon. all_position_importance.tsv - Mean decrease gini for each position for each amino acid as produced by the random forest analysis. combined_sprinzl_data_RF.tsv - Raw input data for features used in the creation of the random forest model. model_testing_accuracy.tsv - Accuracy of the random forest models based on withheld testing data oob_by_aa.tsv - Out of the box error for each machine learning model as calculated per amino acid oob_by_aa_by_order.tsv - Out of the box error for each machine learning model as calculated per taxonomic order. sample_R_file_for_ML.R - R code used to generate random forest models. One script was created for each amino acid, only differing by the amino acid analyzed. Generalist_Specialist_Table_names_10_22.NO_HAPLOMERGER.txt - File used for naming convention changes from y1000+ project. RandomForest.README - Readme file for Random Forest analysis. tRNA_sprinz_one.VAL.R - Code for generating Sprinzl alignment for valine codon generate_single_seq_analysis.sh - Bash script for running Sprinzl alignment pipeline. remove_C.sh - Script used to remove non-Sprinzl positions denoted with a C in the alignment. tRNA_sprinz_one.ArgGCG.R - Code for generating Sprinzl alignment for Arginine GCG. tRNA_sprinz_one.v2.R - Code for generating Sprinzl alignment for all remaining codons. saccharomycotina.RNA.sprinz.fa - Saccharomycotina reference sequences in fasta format tRNA_viz_sprinz.xlsx - Reference Sprinzl alignments from tRNAViz Sprinzl_Alignment.README - Readme for execution of Sprinzl Alignment pipeline