Separable functions of the fission yeast Spt5 carboxyl-terminal domain (CTD) in capping enzyme binding and transcription elongation overlap with those of the RNA polymerase II CTD Journal Article

Authors: Schneider, S.; Pei, Y.; Shuman, S.; Schwer, B.
Article Title: Separable functions of the fission yeast Spt5 carboxyl-terminal domain (CTD) in capping enzyme binding and transcription elongation overlap with those of the RNA polymerase II CTD
Abstract: An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence 1TPAWNSGSK9. The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-ΔCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Keywords: unclassified drug; mutation; nonhuman; protein domain; protein analysis; phenotype; chromosomal proteins, non-histone; cell viability; cell structure; carboxy terminal sequence; enzyme activity; structure activity relation; amino acid sequence; protein structure, tertiary; cell shape; rna polymerase ii; fungus growth; rna precursors; schizosaccharomyces; schizosaccharomyces pombe proteins; schizosaccharomycetaceae; rna processing, post-transcriptional; protein spt5; schizosaccharomyces pombe; transcriptional elongation factors; uracil; transcription elongation factor; cold sensitivity; temperature sensitive clone; antimetabolites
Journal Title: Molecular and Cellular Biology
Volume: 30
Issue: 10
ISSN: 0270-7306
Publisher: American Society for Microbiology  
Date Published: 2010-05-01
Start Page: 2353
End Page: 2364
Language: English
DOI: 10.1128/mcb.00116-10
PUBMED: 20231361
PROVIDER: scopus
PMCID: PMC2863715
Notes: --- - "Cited By (since 1996): 2" - "Export Date: 20 April 2011" - "CODEN: MCEBD" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Yi Pei
    14 Pei
  2. Stewart H Shuman
    529 Shuman