The Ser7 phosphorylation of RNA polymerase II-CTD is required for the recruitment of E3 ubiquitin ligase Asr1 and subtelomeric gene silencing

The carboxy terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is composed of a tandem heptad sequence of Tyr1Ser2Pro3Thr4Ser5Pro6Ser7, which helps facilitate the transcription of all mRNA and the majority of noncoding RNA. The serines of RNAPII-CTD undergo differential phosphorylation, with Ser5 phosphorylation being predominant at the 5' end, Ser2 phosphorylation toward the 3' end, and Ser7 phosphorylation (Ser7P) present throughout the ORF during transcription. The phosphorylation of Ser2 and Ser5 coordinates the recruitment of proteins involved in the progression of transcription. The Ser7P has been shown to play a role in the processing and termination of small nuclear RNA transcription in both budding yeast and humans. Nevertheless, the effect of this phosphorylation mark on protein-coding genes remains unclear. This is despite the fact that substitution of Ser7 with phosphomimetic Glu does not support growth, and highly transcribed mRNA genes show high levels of this phosphorylation mark. In this study, we demonstrate that the interaction between E3 ubiquitin ligase Asr1 and RNAPII is influenced by the Ser7P in both in vitro and in vivo conditions. Asr1 appears to interact with the CTD in a distinct manner, where Ser7 is phosphorylated in the first heptad and Ser5 in the third heptad, involving key residues, such as Lys43, Arg48, Arg168, and Arg252. The Ser7P is important for the recruitment of Asr1 to RNAPII, and Ser7 mutation leads to the upregulation of subtelomeric genes. Ubc2 has been identified as the canonical ubiquitin-conjugating enzyme associated with Asr1.