Figure 2.

Yeast yUtp23 contacts the internal and 3΄ terminal hairpins of the snR30 snoRNA. (A) Pie chart of snoRNA reads from yUtp23 crosslinking data sets. The top 20 hits from two combined yUtp23 data sets (see Supplementary Figure S4B for more detail) are represented as percentages of all reads mapped to snoRNAs. Significant and reproducibly enriched RNAs with essential, non-modifying roles in 18S maturation (snR30, U3 and snR10) are highlighted. (B) Electromobility shift assay (EMSA) showing the binding of GST-yUtp23 or GST (1000 nM, left) or GST-yUtp23 (100–1000 nM, right) to in vitro transcribed radiolabeled snR30 RNA. Free RNA and RNP complexes were separated on 4% native polyacrylamide gels and visualized using a PhoshorImager. (C) yUtp23 crosslinking profile on the snR30 snoRNA. Hits (blue): total reads; deletions (black; dels): mutations and microdeletions representing precise binding sites. Prominent microdeletion peaks in the internal hairpin (IH) and 3΄ terminal hairpin (3΄ HP) are labeled. The positions of the H and ACA boxes within the snR30 RNA are indicated. (D) Predicted secondary structure of the snR30 snoRNA in S. cerevisiae (adapted from (18)). yUtp23 crosslinking sites are marked on the sequence and grey shades indicate peak height. Predominant microdeletion peaks (see panel C) are highlighted by shaded blue circles. The H and ACA boxes are shown in black. (E) A model of the interaction between the 18S ES6 binding sites (m1 and m2) in the 3΄ terminal hairpin (3΄ HP) of snR30 (black) and the 18S ES6 region (nt 801–841, grey). yUtp23 reads (grey) and microdeletion sites in the 3΄ HP (light and dark blue circles) are indicated. Shades of blue represent peak height (see panel C). The ACA box is highlighted in black. (F) EMSA showing the binding of GST-yUtp23 (1300 nM) to full-length (FL) snR30 or snR30 RNA fragments. Free RNA and RNP complexes were analyzed as in panel B. IH: internal hairpin; FL ΔIH: full-length snR30 lacking the internal hairpin.