title: Structural and functional characterization of the N terminus of Schizosaccharomyces pombe Cwf10.
abstract: The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA, yielding mature message. Schizosaccharomyces pombe Cwf10 (homolog of Saccharomyces cerevisiae Snu114 and human U5-116K), an integral member of the U5 snRNP, is a GTPase that has multiple roles within the splicing cycle. Cwf10/Snu114 family members are highly homologous to eukaryotic translation elongation factor EF2, and they contain a conserved N-terminal extension (NTE) to the EF2-like portion, predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation and second-step catalysis. Characterization of Cwf10-NTE by various biophysical techniques shows that in solution the NTE contains regions of both structure and disorder. The first 23 highly conserved amino acids of the NTE are essential for its role in splicing but when overexpressed are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-ΔNTE cells. When the entire NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of independently incorporating into the spliceosome and improving splicing function, possibly indicating a role for the NTE in stabilizing conformational rearrangements during a splice cycle.
authors.affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
authors.forename: S Brent
authors.initials: SB
authors.lastname: Livesay
authors.forename: Scott E
authors.initials: SE
authors.lastname: Collier
authors.forename: Danny A
authors.initials: DA
authors.lastname: Bitton
authors.forename: Jürg
authors.initials: J
authors.lastname: Bähler
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Ohi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism
chemical_list: D017415:Ribonucleoprotein, U5 Small Nuclear; D029702:Schizosaccharomyces pombe Proteins; C000589017:Cwf10 protein, S pombe; D020558:GTP Phosphohydrolases