Identification of queuosine-modified RNAs in S. pombe using metabolic labelling with preQ1-L1 (Q-RIP-Seq)

In most eukaryotes and bacteria, queuosine (Q) replaces the guanosine at the wobble position of tRNAs harboring a GUN anticodon. To investigate whether other RNAs are also Q-or preQ1-modified in Schizosaccharomyces pombe, Q-RIP-Seq was established and applied to RNAs from WT (AEP1) and qtr2? cells (AEP288). Metabolic labeling of RNAs with azido-propyl-preQ1 (preQ1-L1), chemical clicking of a biotin-alkyne followed by immunoprecipitation on streptavidin beads and sequencing of enriched RNAs in WT compared to qtr2? allowed identification of Q-modified RNAs. Overall design: S. pombe cells were metabolically labelled with azido-propyl-preQ1 (preQ1-L1) to label Q-modified RNAs. RNA was extracted, chemically clocked with biotin-alkyne; labeled RNAs were enriched on streptavidin beads and subjected to HTS. HTS reads were analyzed for RNAs enriched in wt compared to qtr2? cells (n = 3).