The carboxyl-terminal domain (CTD) of RNA polymerase II consists of tandem repeats of heptapeptide Y¹S²P³T⁴S⁵P⁶S⁷. The CTD recruits proteins that drive or regulate gene expression. The trafficking of CTD-interacting proteins is orchestrated by remodeling CTD primary structure via Ser/Thr/Tyr phosphorylation and proline cis-trans isomerization, which collectively inscribe a CTD code. The fission yeast CTD consists of 29 repeats. To decipher the output of the fission yeast CTD code, we manipulated CTD length and amino acid content and gauged effects of these changes on gene expression. Whereas deleting 11 heptads has no effect on yeast growth, RNA-seq revealed that 25% of protein-coding transcripts were dysregulated. We profiled the transcriptomes of full-length CTD mutants, in which: all Tyr1 residues were replaced by Phe; all Ser2, Thr4, or Ser7 positions were changed to Ala; and half of the essential CTD code “letters” Pro3, Ser5, and Pro6 were mutated to Ala. Overlapping RNA-seq profiles suggested that a quarter of the up-regulated mRNAs and half of the down-regulated mRNAs seen in full-length CTD mutants might be attributable to a decrement in wild-type heptad number. Concordant mutant-specific profiles were observed for Y1F, S2A, and T4A cells, and for P6•P6A and S5•S5A cells, suggesting that Tyr1–Ser2–Thr4 and Ser5–Pro6 comprise distinct “words” in the CTD code. The phosphate regulon, which is repressed by lncRNA-mediated transcription interference, is de-repressed by CTD mutations P6•P6A and S5•S5A. De-repression of pho1 in P6•P6A and S5•S5A cells depends on cleavage and polyadenylation factor subunits Swd22 and Ppn1 and termination factor Rhn1, signifying that Pro6 and Ser5 mutations elicit precocious lncRNA 3′-processing/termination.

中文翻译：

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裂殖酵母 RNA 聚合酶 II CTD 突变体的转录谱


RNA 聚合酶 II 的羧基末端结构域 (CTD) 由七肽 Y ¹ S ² P ³ T ⁴ S ⁵ P ⁶ S ⁷的串联重复组成。 CTD 招募驱动或调节基因表达的蛋白质。 CTD 相互作用蛋白的运输是通过 Ser/Thr/Tyr 磷酸化和脯氨酸顺反异构化重塑 CTD 一级结构来协调的，它们共同记录了 CTD 代码。裂殖酵母 CTD 由 29 个重复组成。为了破译裂殖酵母 CTD 代码的输出，我们操纵了 CTD 长度和氨基酸含量，并测量了这些变化对基因表达的影响。虽然删除 11 个七肽对酵母生长没有影响，但 RNA 测序显示 25% 的蛋白质编码转录本失调。我们对全长 CTD 突变体的转录组进行了分析，其中： 所有 Tyr1 残基均被 Phe 取代；所有 Ser2、Thr4 或 Ser7 位置均更改为 Ala；一半的必需 CTD 代码“字母”Pro3、Ser5 和 Pro6 突变为 Ala。重叠的 RNA-seq 图谱表明，在全长 CTD 中观察到四分之一的上调 mRNA 和一半的下调 mRNA突变体可能归因于野生型七肽数量的减少。在Y1F 、 S2A和T4A细胞以及P6•P6A和S5•S5A细胞中观察到一致的突变体特异性谱，表明 Tyr1–Ser2–Thr4 和 Ser5–Pro6 在 CTD 代码中包含不同的“单词”。磷酸盐调节子受到 lncRNA 介导的转录干扰的抑制，而 CTD 突变P6•P6A和S5•S5A 则解除抑制。P6•P6A和S5•S5A细胞中pho1的去抑制依赖于切割和多聚腺苷酸化因子亚基 Swd22 和 Ppn1 以及终止因子 Rhn1，这表明 Pro6 和 Ser5 突变引发早熟的 lncRNA 3' 加工/终止。