title: The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function.
abstract: In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the beta-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between beta strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.
authors.affiliation: Program in Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Lindsey-Boltz
authors.forename: G
authors.initials: G
authors.lastname: Chawla
authors.forename: N
authors.initials: N
authors.lastname: Srinivasan
authors.forename: U
authors.initials: U
authors.lastname: Vijayraghavan
authors.forename: M A
authors.initials: MA
authors.lastname: Garcia-Blanco
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012322:RNA Precursors / Q000378:metabolism; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid
chemical_list: C093570:CDC40 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D019913:Nuclear Localization Signals; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins