A Jab1/MPN domain near the C terminus of Prp8p. (A) The 2413-residue S. cerevisiae Prp8p protein carries a Jab1/MPN domain between amino acids 2178 and 2310. A recombinant C-terminal Prp8p fragment containing the Jab1/MPN domain (see below) is depicted underneath the full-length protein. (B) Alignment of the Jab1/MPN domains of several Prp8p orthologs. Residues identical to the human sequence are represented by dashes. The positions that correspond to the JAMM motif (E-X_n-H-X-H-X₇-S-X₂-D) in some other Jab1/MPN domains (Cope et al. 2002; Maytal-Kivity et al. 2002; Verma et al. 2002; Yao and Cohen 2002) are shaded in gray, and those JAMM motif residues that coordinate a Zn²⁺ ion (Tran et al. 2003; Ambroggio et al. 2004) are denoted with asterisks. A 65% Prp8 consensus and the representative Jab1/MPN domain from the NCBI database are aligned underneath the individual Prp8 sequences, and residues in the representative domain that match the Prp8 consensus are underlined. The site of a temperature-sensitive missense (I to N) mutation in S. cerevisiae prp8-28 (van Nues and Beggs 2001) is indicated by an arrow.

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.

Alanine mutations in the noncanonical JAMM residues in PRP8 result in a temperature-sensitive growth phenotype. (A) The indicated strains were grown in complete media at 25°C up to mid-log phase, and serial dilutions of an equal number of cells were spotted onto YPD plates. Cells were grown at 25°C or 37°C and analyzed after 2 d. (B) Alanine mutations in the variant JAMM residues of the Prp8p Jab1/MPN domain do not significantly destabilize the protein at the restrictive temperature in vivo. The indicated strains were grown at 30°C and then shifted to 37°C for 4 h as indicated at the top of each lane. Equivalent amounts of whole-cell extract were resolved by 10% SDS-PAGE and immunoblotted with rabbit α-Prp8p antibodies (Collins and Guthrie 1999). As a loading control, the same blot was probed with α-Npl3p antibodies (Siebel and Guthrie 1996). The smeariness of the Prp8p is due to its susceptibility to endogenous proteases. The mutant Prp8 proteins do not differ significantly from the wild type. (C) Mutations in the Prp8p Jab1/MPN domain reduce splicing efficiency in vivo. Total yeast RNA was prepared from the indicated strains grown as described above in B. A ³²P-radiolabeled oligonucleotide complementary to the U3 snoRNA was hybridized to 12.5 μg of total RNA and extended with M-MLV reverse transcriptase. The reaction products were resolved by denaturing gel electrophoresis and quantified with a PhosphorImager.

Certain mutations in the Jab1/MPN result in reduced levels of U4/U5/U6 tri-snRNP. Splicing extracts from mutant and wild-type strains were incubated at 25°C in the presence or the absence of 2 mM ATP. The reactions were resolved by native polyacrylamide gel electrophoresis, transferred to Hybond-N+ membranes, and sequentially probed for U6, U5, and U4 snRNAs. The position of U4/U5/U6 tri-snRNP, U4/U6 di-snRNP, and the free snRNP complexes are indicated to the left. The rightmost lane in all three panels was loaded on the left side of the gel and then repositioned using Adobe Photoshop. All digital rendering was done to the entire file prior to cutting and pasting that lane.

Prp8p binds ubiquitin in vitro. (A) Lysate from a yeast strain expressing triple HA-tagged Prp8p was prepared and incubated with ubiquitin-Sepharose or Sepharose beads in the presence of indicated amounts of free ubiquitin. The beads were washed, and the bound material was fractionated by SDS-PAGE and immunoblotted with αHA antibody. For comparison, unfractionated extract corresponding to 5% of the input material was analyzed in parallel. (B) Prp8p binds directly to ubiquitin in vitro. Recombinant Prp8p fragment corresponding to C-terminal residues 2143–2413 (including the Jab1/MPN domain) and C-terminal hexahistidine and HA tags was expressed and purified. The ability of this fragment to bind ubiquitin in vitro was analyzed as in A. (C) The Jab1/MPN domain of Prp8p is important for ubiquitin binding. Untagged recombinant Prp8p fragment that contained a wild-type or mutated Jab1/MPN domain (corresponding to the prp8-602 and prp8-603 alleles) was expressed in Escherichia coli. Ubiquitin binding was analyzed as in A, except that the binding was done at room temperature or 37°C and the immunoblots were probed with α-Prp8p antisera (Collins and Guthrie 1999). (D) Mutations in Prp8p that do not affect ubiquitin binding. Untagged recombinant Prp8p fragment that contained a wild-type or mutated Jab1/MPN domain (corresponding to the prp8-1, prp8-28, and prp8-D144 alleles) was expressed in E. coli. Ubiquitin binding was analyzed as in A, except that the binding was done at room temperature or 37°C and the immunoblots were probed with α-Prp8p antisera (Collins and Guthrie 1999). (E) An I44A mutation in ubiquitin diminishes ubiquitin binding by Prp8p fragment. Untagged recombinant Prp8p fragment that contained a wild-type Jab1/MPN domain was expressed in E. coli. Ubiquitin binding was analyzed as in A, except that binding was done with hexahistidine-tagged wild-type or I44A ubiquitin that was immobilized on Ni-NTA magnetic agarose resin and the immunoblots were probed with α-Prp8p antisera (Collins and Guthrie 1999).

Quantitative analysis of Prp8p/ubiquitin binding by BIA-core surface plasmon resonance. (A) Various concentrations of purified Prp8p fragment were injected over hexahistidine-tagged ubiquitin that was immobilized on a Ni²⁺-nitrilotriacetic acid (Ni-NTA) sensor chip. Prp8p binding was done at 4°C and was recorded by the instrument as “response units” that were normalized for the ubiquitin density on the chip. (B) Data from A were corrected for background and fit to a binding isotherm (assuming 1:1 stoichiometry) using KaleidaGraph (Synergy) software. The fit indicated an equilibrium dissociation constant (K_d) of 380 ± 70 μM.