Combining Nanopore direct RNA sequencing with genetics and mass spectrometry for analysis of T-loop base modifications across 42 yeast tRNA isoacceptors.

ABSTRACT: Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits-the potent influence

SUBMITTER: Shaw EA 

PROVIDER: S-EPMC11514469 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature