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Background: Psh1 is an E3 ubiquitin ligase that controls CenH3/Cse4 levels through proteolysis in budding yeast. Results: Psh1 is phosphorylated in vivo by CK2, and its E3 ligase activity is promoted. Conclusion: Phosphorylation is crucial in Psh1-assisted control of Cse4 levels, and the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation. Significance: This work reports a previously unknown function of CK2 in CenH3/Cse4 regulation., Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2Δ strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2Δ strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.

Messenger RNA processing is coupled to RNA Polymerase II (RNAPII) transcription through coordinated recruitment of accessory proteins to the Rpb1 C-terminal domain (CTD). Dynamic changes in CTD phosphorylation during transcription elongation are responsible for their recruitment, with serine 5 phosphorylation (S5-P) occurring towards the 5’ end of genes and serine 2 phosphorylation (S2-P) occurring towards the 3’ end. The proteins responsible for regulation of the transition state between S5-P and S2-P CTD remain elusive. We show that a conserved protein of unknown function, Rtr1, localizes within coding regions, with maximum levels of enrichment occurring between the peaks of S5-P and S2-P RNAPII. Upon deletion of Rtr1, the S5-P form of RNAPII accumulates in both whole cell extracts and throughout coding regions; additionally, RNAPII transcription is decreased and termination defects are observed. Functional characterization of Rtr1 reveals its role as a CTD phosphatase essential for the S5- to S2- P transition.