Structural and functional analysis of essential pre-mRNA splicing factor Prp19p.
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.
Ohi
Melanie D
MD
Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA.
Vander Kooi
Craig W
CW
Rosenberg
Joshua A
JA
Ren
Liping
L
Hirsch
Justin P
JP
Chazin
Walter J
WJ
Walz
Thomas
T
Gould
Kathleen L
KL
D018797:Cell Cycle Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D019281:Dimerization; D005838:Genotype; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D047468:Immunoprecipitation; D008854:Microscopy, Electron; D008954:Models, Biological; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins; D017381:Spliceosomes / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D020798:Two-Hybrid System Techniques; D014461:Ultracentrifugation
C513239:CEF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; C079877:PRP19 protein, S cerevisiae; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C085875:cdc5 protein, S pombe