Enzymatic Probing of RNA Structure

5′-end labeled transcripts were prepared as described for the EMSAs. The enzymatic probing was carried out as previously described (Sievers et al., 2014 (link)), with some deviations. Briefly, for the alkaline hydrolysis ladder, 0.2 pmol of labeled RNA was mixed with alkaline hydrolysis buffer (Ambion) and 10 μg of yeast tRNA (Ambion) in a total volume of 10 μL and incubated at 95°C for 5 min; for T1 control sample, 0.2 pmol of labeled RNA was denatured and incubated with 0.01 U of T1 RNase (Ambion) for 5 min. Structure probing RNA interactions were incubated at 37°C for 1 h before treating the samples with the indicated cleaving agent: 0.01 U T1 RNase for 5 min and 0.0015 U V1 RNase (Ambion) for 2 min. Control samples were prepared likewise (except for the cleaving agents) and incubated at 37°C for the duration of the experiment. Samples were placed on ice and mixed with 2× loading buffer type II (Ambion). Five μL of each sample was separated on an 8% denaturing polyacrylamide gel. RNA bands were visualized and analyzed as described for the northern blotting experiments.