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.
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 (1)TPAWNSGSK(9). 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-DeltaCTD 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.
Schneider
Susanne
S
Graduate Program in Molecular Biology, Weill Cornell Medical College, New York, New York 10065, USA.
Pei
Yi
Y
Shuman
Stewart
S
Schwer
Beate
B
D000595:Amino Acid Sequence; D000963:Antimetabolites / Q000378:metabolism; D048430:Cell Shape; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014498:Uracil / Q000031:analogs & derivatives / Q000378:metabolism
D000963:Antimetabolites; D002868:Chromosomal Proteins, Non-Histone; D012322:RNA Precursors; D029702:Schizosaccharomyces pombe Proteins; C549622:Spt5 protein, S pombe; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D014498:Uracil; D012319:RNA Polymerase II; C100248:azauracil