Graduate Program in Molecular Biology, Weill Cornell Medical College, New York, New York 10065, USA.
Mol Cell Biol. 2010 May;30(10):2353-64. doi: 10.1128/MCB.00116-10. Epub 2010 Mar 15.
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.
一个连接 mRNA 加帽酶、RNA 聚合酶 II(Pol II)羧基末端结构域(CTD)、延伸因子 Spt5 以及 Cdk7 和 Cdk9 蛋白激酶的相互作用网络被认为构成了转录延伸检查点。这个网络的关键是 Spt5,它调节早期转录延伸,并通过与加帽酶的物理结合在 pre-mRNA 加工中发挥作用。裂殖酵母 Spt5 的 CTD 由串联的非肽重复序列组成,共有 9 个氨基酸,序列为(1)TPAWNSGSK(9)。Spt5 CTD 结合加帽酶,是 Cdk9 激酶的苏氨酸磷酸化的底物。在这里,我们报道 S. pombe Spt5 CTD 的缺失导致生长缓慢和细胞形态异常。Pol II CTD 截短会加剧 spt5-DeltaCTD 表型的严重程度,而过表达加帽酶 RNA 三磷酸酶和 RNA 鸟苷转移酶则会改善这种表型。这些结果表明,Spt5 和 Pol II CTD 在招募加帽酶方面发挥了功能重叠的作用。我们通过对 Spt5 CTD 的保守九肽进行丙氨酸扫描来探究其结构-活性关系。T1A 突变消除了 Cdk9 对 CTD 的磷酸化,但不影响 CTD 与加帽酶的结合。T1A 和 P2A 突变引起冷敏感(cs)和温度敏感(ts)生长缺陷,并对 6-氮杂尿嘧啶的生长抑制敏感,这种敏感性因 Pol II CTD 的部分截短而加剧。T1A 表型可以被磷酸模拟 T1E 突变挽救,但不能被加帽酶过表达挽救。这些结果表明,在裂殖酵母中,Spt5 CTD 磷酸化在 Pol II 转录延伸中发挥了积极作用,与它与加帽酶的相互作用不同。携带 Spt5 CTD T1A 和 Pol II CTD S2A 突变的酵母细胞的存活表明,Cdk9 激酶除了 Spt5 和 Pol II CTD-Ser2 之外,还有一个必需的靶标。