Detection of Q-modification in RNAs from S. pombe and S. flexneri via reverse transcription using RT-KTq I614Y (Q-MaP-Seq)

In most eukaryotes and bacteria, queuosine (Q) replaces the guanosine at the wobble position of tRNAs harboring a GUN anticodon. To faithfully detect Q-modification in RNAs from Schizosaccharomyces pombe and Shigella flexneri, Q-MaP-Seq was established and applied to tRNAs from S. pombe WT (AEP1) cells and Shigella flexneri WT cells and tgt∆ cells. Q-modification of in vitro-transcribed RNAs and RNAs isolated from S. pombe and S. flexneri followed by reverse transcription using the RT-active DNA polymerase variant RT-KTq I614Y and sequencing of unmodified compared to modified RNAs allowed identification of Q-sites within tRNAs. in vitro-trasncribed tRNAs Asn and Asp were modified with queuine. Furthermore, RNA from S. pombe cells and S. flexneri cells were Q-modified in vivo. RNA from cells was extracted, and reverse transcribed using the RT-KTq I614Y; tRNAs were amplified and subjected to HTS. HTS reads were analyzed for error RT-signatures in Q-modified compared to unmodified tRNAs.