Creation and characterization of a yeast HSH155 allele humanized for sensitivity to splicing inhibitors. (A) Sequence alignment of the BP-A binding pockets of HSH155 (top line) and SF3B1 (middle line). HRs 15 and 16 are shown with their paired α helices in blue above the alignment. The bottom line shows the sequence of hsh155-ds with blue boxes indicating the yeast-specific amino acids, and gold boxes indicating the human-specific amino acids. Amino acids identical in yeast and humans are unshaded. The amino acid numbers are shown above (yeast) and below (human). Below the sequence is a model of SF3B1 (left) or just the HRs 15 and 16 (right) bound to Plad-B (black, Cretu et al. 2018, PDB: 6EN4). Human-specific amino acids replaced in the yeast protein are shown in gold. (B) Serial dilution of WT HSH155 and hsh155-ds yeast strains on YPD plates to test for temperature sensitivity. (C) Western blot of strains expressing wild-type and hsh155-ds tagged with GFP. Lane 1, markers; lane 2, WT Hsh155 carboxy-terminally tagged with GFP; lane 3, hsh155-ds tagged with GFP; lane 4, untagged wild-type Hsh155; lane 5, positive control SIR1 tagged with GFP. The blot was probed with mouse anti-GFP antibodies, visualized using secondary goat anti-mouse antibody, then scanned on a Li-Cor infrared scanner. (D) Measurement of Plad-B sensitivity by RT-PCR of the MATa1 first intron. Samples were taken at times after the addition of DMSO (top) or Plad-B to 5 µM (bottom) at 0, 1, 4, and 8 min. Positions of the PCR product derived from unspliced RNA (US) and spliced RNA (S) are indicated by the labels.
