18S rRNA modification under normal and high temperature conditions in yeast (A,C) and amphibian cells (D,E). (A) Yeast optimized SNORA18 (C-to-U mutant, depicted in Fig. 2A) does not induce pseudouridylation of yeast 18S rRNA at position 1656 (equivalent to human 18S-1720) in the BY4741 strain grown at 30°C, nor can it do so at 37°C (blue traces). “Perfect” SNORA18, a “perfect guide” RNA for position 1656 in yeast 18S rRNA made from SNORA18 (depicted in Fig. 2A), induces pseudouridylation of the target position both at 30°C (not shown) and 37°C (top pink trace, star). Note there is no Pus7p activity on position 1585 in 18S rRNA both at 30°C and 37°C. Positions that were expected to be modified are indicated with arrowheads. (B) Postulated base-pairing of X. tropicalis and X. laevis SNORA28 with 18S rRNA. For the 5′-terminal (left schematic) and the 3′-terminal pseudouridylation pockets (right schematic), target positions and corresponding modifications are indicated on the right side of each pocket. A mismatched nucleotide in the 3′-terminal X. laevis ASE is highlighted in green. (C) Yeast cells grown at 37°C do not induce pseudouridylation activity of X. laevis SNORA28 on yeast 18S rRNA at position 808 (equivalent to Xenopus 18S-828). Note the increased modification level at position 759, the other target of SNORA28 (light blue trace), as compared to controls (gray and red traces). X. tropicalis SNORA28 is functional on position 808 (top dark blue trace, star). (D,E) X. laevis XTC cells (D) and axolotl embryos (E) grown at elevated temperatures do not have altered pseudouridylation patterns of 18S rRNA. (D) When snoRNA with a functional pseudouridylation pocket for 18S-828 (xtSNORA28 > U2-Ψ60, blue trace) is expressed in XTC cells, this position is modified (star). Unmodified position 828 is indicated with arrowheads.
