Sub1 contacts the RNA polymerase II stalk to modulate mRNA synthesis

Abstract

Biogenesis of messenger RNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II (RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions and Sub1 globally influences CTD phosphorylation, though its mechanism remains mostly unknown. Here, we show that Sub1 physically interacts with the RNAPII stalk domain, Rpb4/7, likely through its C-terminal region, and associates with Fcp1. While Rpb4 is not required for Sub1 interaction with RNAPII complex, a fully functional heterodimer is required for Sub1 association to promoters. We also demonstrate that a complete CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Finally, genetic data show a functional relationship between Sub1 and the RNAPII clamp domain. Altogether, our results indicate that Sub1, Rpb4/7 and Fcp1 interaction modulates CTD phosphorylation. In addition, Sub1 interaction with Rpb4/7 can also modulate transcription start site selection and transcription elongation rate likely by influencing the clamp function.

Sub1 and RNAPII, until termination does them part

“…Supporting this idea, we identified a specific genetic interaction between sub1ΔCT and rpb4Δ , but not with a mutation altering Sub1 DNA binding, and showed that the association of Sub1ΔCT with gene promoters is significantly reduced in the absence of RPB4 . 21 This is the first evidence for a role of the Sub1-CT region.

Figure 1.

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“… 27,41 Regardless, our studies suggest the CT region is necessary for the stability of Sub1 and it could regulate its DNA binding capacity, while interacting with Rpb4/7. 21 …”

“…However, a fully functional Rpb4/7 heterodimer is necessary for Sub1 to stably associate with chromatin after RNAPII recruitment to the PIC. 21 We have proposed a model, where Sub1 is recruited to RNAPII through interaction with Rpb4/7, TFIIB, and DNA. 12-14,16 One hypothesis is that once at the PIC, Sub1 interacts with Rpb4/7 via its CT region to keep associated to RNAPII and chromatin ( Fig.…”

“… Schematic model showing the hypothetical localization of Sub1 during transcription initiation. Sub1 is bound to the promoter by interacting with upstream DNA at the junction between single- and double-stranded DNA 14 through its DNA Binding Domain (Sub1 DBD) 21 . The proposed localization of Sub1 in this model explains the reported physical and genetic interaction of Sub1 with TFIIB 13,55 , as well with TFIIE and TFIIH.…”

“… 16,19,20 More recently, we have shown that Sub1 directly interacts with the RNAPII stalk domain formed by the Rpb4 and Rpb7 subunits, and is genetically related to the polymerase clamp domain. 21 This may help to explain how this multifaceted factor may influence different processes during the whole transcription cycle in addition to globally modulating Rpb1-CTD phosphorylation. 18 Thus, in the course of the transcription cycle, Sub1 could exert its function at different stages through the association with specific factors that directly interact with Rpb4/7 and/or the clamp domain.…”