Deletion of the region containing the Prp8p Jab1/MPN domain is lethal in S. cerevisiae. (A) The Jab1/MPN domain (amino acids 2178–2310) was deleted from the PRP8 gene on a HIS3-marked plasmid (pRS313-prp8Δjab1/mpn-3HA). Strains harboring pRS313-prp8Δjab1/mpn-3HA as well as a wild-type PRP8 gene on a URA3-marked plasmid (Kuhn et al. 1999) were grown at 25°C to mid-log phase in medium lacking histidine and uracil. Serial dilutions of an equal number of cells were spotted onto 5-FOA plates. Cells were grown at room temperature and analyzed after 3–4 d. (B) The Jab1/MPN domain in Prp8p is required for stable protein expression. Splicing extracts were prepared from the strains indicated in A, following growth in media lacking uracil and histidine. The PRP8 allele on the HIS3-marked plasmid also contained a triple-HA tag at the C terminus. Equal amounts of protein were loaded on an SDS-PAGE gel and immunoblotted with an antibody against the triple-HA tag. As a loading control, the same blot was probed with α-Rsp5p antibody.
