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. 2011 Feb 23;39(12):5164–5180. doi: 10.1093/nar/gkr044

Figure 4.

Figure 4.

Expression of U3 snoRNAs mutated in nucleotides that base pair changes the reactivity of 5′-ETS nucleotides to DMS in vivo. Total RNA was extracted from yeast cells depleted of the endogenous U3 snoRNA as described in the legend to Figure 2. Yeast contained either a plasmid expressing no U3 snoRNA (empty vector; EV), U3 snoRNA wild-type (U3 WT), a U3 snoRNA with the 5′-hinge mutated (U3 5H) (17) or a U3 snoRNA with the Box A mutated (U3 Box A) (33). (A) Expression of U3 snoRNA mutants from plasmids was detected via their unique sequence tag by northern blotting (oligo SD13) (15), while another oligonucleotide was used to visualize all forms of U3 snoRNA (SD74). The U14 snoRNA was detected with a specific oligonucleotide. (B) rRNA levels analyzed by ethidium bromide staining on agarose gels. (C–E) In vivo DMS modification of the RNA at 16 h after U3 snoRNA depletion was carried out in cells containing the plasmids described in (A). Total RNA was extracted from unmodified and modified cells and analyzed by primer extension with the following oligonucleotides: (C) 400-5′-ETS; (D) 611-5′-ETS; (E) 693-5′-ETS. Lanes A and G are dideoxy sequencing lanes. The nucleotide numbers from the start site of transcription are indicated on the left side of the gels. The lines on the right side of the gels highlight regions were nucleotides with changed reactivity to DMS are present. Nucleotides with altered reactivity as a result of the expression of the mutated U3 snoRNAs depicted on a portion (nucleotides 260–700) of the secondary structure of the 5′-ETS: (F) U3 5H and (G) U3 Box A. Blue circles denote nucleotides with lower reactivity, while red circles denote nucleotides with higher reactivity when the U3 snoRNA mutants are expressed, using nucleotide reactivity when no U3 snoRNA is expressed (EV) as a reference point. The size of the circles represents the intensity of the change. The purple boxes highlight the nucleotides of the 5′-ETS that were previously known to base pair the U3 snoRNA, the green boxes highlight the nucleotides involved in the novel base pairing and the cleavage sites are indicated.