Characterizing the Structure and Function of the N-Terminus of Schizosaccharomyces Pombe Cdc5, a Pre-Mrna Splicing Factor

Abstract

The spliceosome is a dynamic macromolecular machine composed of 5 small nuclear ribonucleoproteins (snRNPs), the Nineteen Complex (NTC), and other proteins that catalyze the removal of introns from pre-mRNA. The NTC is a highly conserved sub-complex of the spliceosome consisting of approximately fifteen proteins and is required for the transition of the spliceosome from an inactive to activated complex. However the mechanism of NTC function in this process is not understood. Schizosaccharomyces pombe Cdc5, a core subunit of the NTC, is an essential protein that contains two highly conserved N-terminal Myb repeats, a N-terminal non-canonical Myb-like repeat (MLR) as well as a less well conserved C-terminus. Both in vivo and in vitro studies have shown that the C-terminus of Cdc5 directly interacts with other NTC components; however, a specific function for the N-terminus of Cdc5 in pre-mRNA splicing has not been determined.Our goal is to understand the specific role(s) of Cdc5 in NTC function and pre-mRNA splicing. Using a combination of yeast genetics, NMR secondary structure analysis, Chemical Shift (CS) Rosetta modeling, and an RNA binding assay we have shown the MLR is structurally distinct from the canonical Myb fold, that the N-terminus of Cdc5 preferentially binds double stranded RNA, and defined the importance the of the N-terminal Cdc5 Myb repeats and MLR in fission yeast.