Computational Identification of Putative Split tRNA Genes with A34 Anticodons in Methanosarcina barkeri Strain Wiesmoor: Implications for Genome Annotation and Translational Biology

Archaeal genomes are known to contain intronless, intron-containing, and split transfer RNA (tRNA) genes; yet identifying all of them remains a challenging task in bioinformatics due to their unusual structures and complex features. Many rare or hidden tRNA genes are often missed by standard computational tools. In this study, we aimed to identify such overlooked tRNA genes by examining whether distant regions of the genome can together form a functional tRNA molecule. To achieve this, we developed a computational method, tRNA search algorithm. This method scans the genome for conserved sequence motifs at the 5′ and 3′ ends and reconstructs possible tRNA structures following the split tRNA concept. The predicted sequences were further analyzed for their cloverleaf secondary structure and characteristic splice-site motifs such as bulge–helix–bulge (BHB). Using this approach, we analyzed the genome of Methanosarcina barkeri strain Wiesmoor (MBSW) and identified several previously undetected split tRNA genes. Notably, we found rare tRNAs with adenine at position 34 of the anticodon and observed unique exon arrangements where a common exon combines with different variable exons to form functional tRNAs. These findings improve genome annotation and enhance understanding of tRNA diversity and evolution.