Testing potential U3 snoRNA–pre-rRNA interactions. (A) Comparison of DMS modifications patterns of total RNA and newly synthesized RNAs. The nucleotide positions are indicated on the side of the gel. A primer extension stop generated by natural modification of C1639 is indicated (1639C-2′-O-methyl). The graph presents the intensity of bands in each lane: (black line) total RNA; (red line) 2 min; (green line) 4 min; (blue line) 6 min of 4TU labeling. (B) DMS modification of RNA present in purified pre-40S complex. The purification was performed using Tsr1-HTP as bait. 18S and 20S rRNAs were recovered as described for panel C. The graph presents the intensity of bands in the lane showing modification of 20S rRNA. Primer 104 (complementary to the apical loop of 18S rRNA helix 44) was used in all experiments. All experiments were repeated twice. (C) Isolation of 18S and 20S rRNA. RNA was extracted from purified pre-40S complexes. DMS modified (+) and unmodified (−) RNA was separated on a 2% agarose, BPTE gel. The gel was stained by SYBR to visual the RNA, the 18S and 20S regions were excised form the cut gel, and RNA was eluted.
