Merging nanopore direct RNA sequencing with molecular genetics and mass spectrometry for analysis of T-loop base modifications across 42 yeast tRNA isoacceptors
Previously we applied Nanopore technology to directly sequence and classify full-length E. coli tRNA strands, including all 43 isoacceptors. Evidence for nucleotide modifications was observed in sequence miscalls when comparing wild type Nanopore reads with their synthetic canonical control counterparts. Given the importance of Nanopore-based measurements, we reasoned that more robust cross-validation is warranted. In this study, we focused on three highly conserved modifications in the T-loop of the 42 known cytosolic tRNA isoacceptors in Saccharomyces cerevisiae, i.e. 5-methyluridine (m5U54), pseudouridine (psi55), and 1-methyladenosine (m1A58). Alignment of Nanopore reads suggested that m5U and m1A modifications are dependent on the U55 being pseudouridylated. To test this, we analyzed modification-specific enzyme knockout genetic strains. Mass spectrometry unambiguously confirmed the absence of specific modifications (e.g. m5U, m1A) for corresponding genetic knockout strains. This combined analysis cross-validated the presence of concerted T-loop modifications in yeast tRNA, consistent with prior reports. We then implemented a classification strategy that could accurately predict the presence or absence of a specific modification (e.g. m1A) based on miscalls in the T-loop of an isoacceptor. We also developed a modified tRNA-adaptation strategy that allowed nanopolish-based ionic current alignments to be performed for tRNA strands. Our results demonstrate how orthogonal technologies (DRS and mass spectrometry), combined with molecular genetics, permit detection of changes in modification profiles among individual, full-length tRNA strands.