Transcriptional profiling of fission yeast RNA polymerase II CTD mutants.
The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol2) consists of tandem repeats of a consensus heptapeptide Y 1  S 2  P 3  T 4  S 5  P 6  S 7  The CTD recruits numerous 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 heptad repeats. To decipher the output of the fission yeast CTD code, we genetically manipulated CTD length and amino acid content and then gauged the effects of these changes on gene expression. Whereas deleting 11 consensus heptads has no obvious effect on fission yeast growth, RNA-seq revealed that 25% of the protein-coding transcripts were dysregulated by CTD truncation. 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 complement of 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 CTD heptad number. Concordant mutant-specific transcriptional 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 fission yeast 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 transcription termination factor Rhn1, signifying that Pro6 and Ser5 mutations elicit precocious lncRNA 3'-processing/termination.
Garg A, Sanchez AM, Schwer B, Shuman S