Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity
Monitoring dynamic changes in cellular tRNA pools is challenging, due to their extensive post-transcriptional modification. A key requirement for any tRNAseq method is the use of a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show that the recently developed group-II intron RT Induro has the processivity and efficiency necessary to profile tRNA dynamics. In a tRNAseq method simpler and more comprehensive than the best methods to data, we show that Induro progressively increases readthrough of tRNA over time and that the mechanism of increase is selective removal of RT stops at modified bases, without altering the misincorporation frequency. We provide parallel datasest of the misincorporation profile of Induro and the related TGIRT RT to facilitate the prediction of non-annotated modifications. We report an unexpected modification profile among human proline isoacceptors, absent from mouse and lower eukaryotes, that indicates new biology in the decoding of proline codons.