Analysis of the Ty3 RNA genome structure under cellular stress

The objective was to provide high-resolution data and comprehensive analysis regarding the impact of cellular stress on the secondary structure of the yeast Ty3 retrotransposon RNA genome using the SHAPE-MaP strategy. To aim this goal the structure of Ty3 gRNA was studied in yeast subjected to selected stress factors - kasugamycin and vanillin (reported previously as translation inhibitors, and additionally, vanillin as a stress granule indicator), and in the native in vivo conditions as a control.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, yeast cell culture in the mid-log phase was treated with selected antibiotics (2.4mM kasugamycin for 20 min/20mM or 50mM vanillin for 30 min). The untreated culture was studied as a control. Then, cells were subjected to modification with 100mM NAI SHAPE reagent for 20 min (NAI-treated samples, Plus) or mock treatment (DMSO-treated samples, Minus). The ex vivo SHAPE-MaP experiment was also performed - total RNA was extracted and refolded before SHAPE probing (100 mM NAI for 15 min). After modification, SHAPE adduct locations were detected using specific primers (two overlapping amplicons covering +1-725 nts and +631-1371 nts of the Ty3 gRNA, respectively) 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 one or two biological replicates, depending on the primary result.