Nick Reiter
Last active: 1/21/2015

Structural and functional insights into the N-terminus of Schizosaccharomyces pombe Cdc5.

Collier SE, Voehler M, Peng D, Ohi R, Gould KL, Reiter NJ, Ohi MD
Biochemistry. 2014 53 (41): 6439-51

PMID: 25263959 · PMCID: PMC4204884 · DOI:10.1021/bi5008639

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.

MeSH Terms (21)

Binding Sites Catalytic Domain Cell Cycle Proteins Gene Deletion Models, Molecular Mutant Proteins Nuclear Magnetic Resonance, Biomolecular Peptide Fragments Protein Conformation Protein Folding Protein Interaction Domains and Motifs Protein Stability Recombinant Proteins RNA, Double-Stranded RNA, Fungal RNA, Small Nuclear RNA-Binding Proteins RNA Splicing Schizosaccharomyces pombe Proteins Spliceosomes Titrimetry

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