title: Structural and functional insights into the N-terminus of Schizosaccharomyces pombe Cdc5.
abstract: The spliceosome is a dynamic macromolecular machine composed of five small nuclear ribonucleoparticles (snRNPs), the NineTeen Complex (NTC), and other proteins that catalyze the removal of introns mature to form the mature message. The NTC, named after its founding member Saccharomyces cerevisiae Prp19, is a conserved spliceosome subcomplex composed of at least nine proteins. During spliceosome assembly, the transition to an active spliceosome correlates with stable binding of the NTC, although the mechanism of NTC function is not understood. Schizosaccharomyces pombe Cdc5, a core subunit of the NTC, is an essential protein required for pre-mRNA splicing. The highly conserved Cdc5 N-terminus contains two canonical Myb (myeloblastosis) repeats (R1 and R2) and a third domain (D3) that was previously classified as a Myb-like repeat. Although the N-terminus of Cdc5 is required for its function, how R1, R2, and D3 each contribute to functionality is unclear. Using a combination of yeast genetics, structural approaches, and RNA binding assays, we show that R1, R2, and D3 are all required for the function of Cdc5 in cells. We also show that the N-terminus of Cdc5 binds RNA in vitro. Structural and functional analyses of Cdc5-D3 show that, while this domain does not adopt a Myb fold, Cdc5-D3 preferentially binds double-stranded RNA. Our data suggest that the Cdc5 N-terminus interacts with RNA structures proposed to be near the catalytic core of the spliceosome.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center , Nashville, Tennessee 37232, United States.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Collier
authors.forename: Markus
authors.initials: M
authors.lastname: Voehler
authors.forename: Dungeng
authors.initials: D
authors.lastname: Peng
authors.forename: Ryoma
authors.initials: R
authors.lastname: Ohi
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Gould
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Reiter
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Ohi
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D012326:RNA Splicing; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017186:Titrimetry
chemical_list: D018797:Cell Cycle Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C085875:cdc5 protein, S pombe