Structural and functional analysis of essential pre-mRNA splicing factor Prp19p.

Ohi MD, Vander Kooi CW, Rosenberg JA, Ren L, Hirsch JP, Chazin WJ, Walz T, Gould KL
Mol Cell Biol. 2005 25 (1): 451-60

PMID: 15601865 · PMCID: PMC538785 · DOI:10.1128/MCB.25.1.451-460.2005

U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome. Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability. Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil. Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p. Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils. These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC.

MeSH Terms (29)

Cell Cycle Proteins Chromatography, Gel Circular Dichroism Dimerization DNA Mutational Analysis Genotype Image Processing, Computer-Assisted Immunoblotting Immunoprecipitation Microscopy, Electron Models, Biological Polymerase Chain Reaction Protein Binding Protein Conformation Protein Structure, Tertiary Recombinant Fusion Proteins RNA, Messenger RNA-Binding Proteins RNA Splicing RNA Splicing Factors Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Schizosaccharomyces Schizosaccharomyces pombe Proteins Spliceosomes Structure-Activity Relationship Temperature Two-Hybrid System Techniques Ultracentrifugation

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