Mapping the structural landscape of the yeast Ty3 retrotransposon RNA genome
The objective was to provide high-resolution data and comprehensive analysis regarding the secondary structure of the yeast Ty3 retrotransposon RNA genome using the SHAPE-MaP strategy. To obtain structural data for an active Ty3 gRNA, we used the Saccharomyces cerevisiae strain BY4741 with Ty3 expressed from the galactose-inducible promoter on a high-copy plasmid. In brief, RNA in different experimental conditions (in vivo, ex vivo, or in vitro) was subjected to modification with SHAPE reagent (NAI-treated samples, Plus) or mock treatment (DMSO-treated samples, Minus). For in vivo SHAPE-MaP experiments, total RNA in yeast was modified with NAI. Otherwise, total RNA was extracted and refolded before SHAPE probing (ex vivo state). Also, SHAPE-MaP experiments were performed for the transcripts (in vitro state) representing Ty3 5' (WT and delPAL6) or 3' end, optionally after Ty3 Ncp9 protein-induced dimerization or tRNAiMet annealing. After modification, SHAPE adduct locations were detected using specific primers (8 overlapping amplicons for Ty3 gRNA) and mutational profiling (MaP), which exploits error-prone reverse transcription. Then, samples were converted into sequencing libraries and sequenced in the PE250 or PE300 Illumina system. Experiments were performed with two biological replicates.