A highly conserved Saccharomyces cerevisiae homolog of hUAP56. (A) Amino acid alignment. The S. cerevisiae yUAP/Sub2p amino acid sequence was aligned to human UAP56 (hUAP) and the hypothetical Schizosaccharomyces pombe UAP (pUAP) found in database searches. Identical amino acids in all three homologs are shaded. Conserved motifs characteristic of DExD/H box protein family are indicated at the bottom of the alignment. Star indicates the mutated aspartic acid residue in sub2-100, a temperature-sensitive allele of SUB2. (B) hUAP56 can functionally substitute for yUAP/Sub2p. Growth of cells on 5FOA, galactose plate is shown.

A temperature-sensitive mutant of yUAP/Sub2p. (A) Growth phenotype of sub2-100 containing strain, MZ306. (WT yUAP/Sub2), yeast strain MZ102b carrying SUB2. (Sub2-100), yeast strain MZ306 carrying a mutant allele of SUB2, sub2–100. (B) Splicing analysis. Splicing extracts were derived from strain MZ102b (lanes 1,2) and MZ306 grown at 30°C (lanes 3,4,5), respectively. Splicing reactions were carried out at 25°C for 30 min. In lane 4, the splicing extract was preincubated at 37°C for 5 min. In lanes 2 and 5, splicing extracts were preincubated at 37°C for 15 min.

Role of DExD/H box protein family consensus motifs in yUAP/Sub2p function. (A) Amino acid sequences of the mutated motifs. Arrows indicate the mutated residues. (B) Effects of yUAP/Sub2p mutations on cell growth. Strains used in top panel were derived from strain MZ102b and contain plasmid pMZ11 (SUB2, URA3, CEN/ARS) and an integrated copy of wild-type yUAP/Sub2p (MZ104) or various yUAP/Sub2p mutants (MZ106, MZ107, MZ108, and MZ109) expressed from the GAL1 promoter. Growth of cells carrying the indicated genes on His⁻ glucose or 5FOA His⁻ galactose is shown. In the bottom panel, W303 was used as the parental strain. Growth of cells on a His⁻ galactose plate is shown.

yUAP/Sub2p is an essential pre-mRNA splicing factor. (A) Schematic diagram of a conditional yUAP/Sub2p expression construct. (B) Growth curve of strain MZ105, which conditionally expresses yUAP/Sub2p. Strain MZ102a, which expresses yUAP/Sub2p constitutively, was used as a control. Cultures of MZ105 and MZ102a in 4% galactose containing medium were centrifuged and the cells resuspended in rich medium containing 4% glucose at time zero. Every 2 h, the OD₆₀₀ of the cultures was measured and the concentration of cells adjusted to remain between OD₆₀₀ 1.0 and 3.0. (C) Splicing analysis. Splicing reactions were carried out at 25°C for 30 min. In lane 1, splicing extract was prepared from strain MZ105 grown in galactose-containing media. In lanes 2–5, splicing extract was prepared from strain MZ105 16 h following shift from galactose- to glucose-containing media. In lanes 3–5, 0.1 μL, 0.5 μ, and 1 μL of GST-yUAP/Sub2p (0.1 mg/mL) was added to the reaction mixture.

yUAP/Sub2p is required for commitment complex formation. (A) Analysis of splicing complex formation using U1 snRNA-, U2 snRNA-, or yUAP/Sub2p-depleted extracts. yUAP/Sub2p-, U1 snRNA-, or U2 snRNA-depleted splicing extract was derived from strain MZ105, BS-Y82, or BS-Y88, respectively, 16 h after shift from galactose- to glucose-containing media. Splicing reactions (40% extract) were carried out and analyzed on a native gel. In lanes 4–6, 20% of the indicated extracts were used. In lanes 7 and 8, extracts were prepared from strain W303 and MZ105 grown in galactose-containing media. Commitment (CC1 and CC2) and spliceosomal (SP) complexes are indicated. (B) Analysis of splicing complex formation using sub2-100 containing extracts. Splicing extracts were derived from strain MZ102b (lanes 1,2), and MZ306 grown at 30°C (lanes 3,4), respectively. Splicing reactions (40% extract) were carried out for 20 min and analyzed on a native gel. In lanes 2 and 4, splicing extracts were preincubated at 37°C for 15 min before assembling the splicing reaction mixture.