RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells.
Bottom Line: Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y).Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro.Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription.
Affiliation: Department of Biological Sciences, Columbia University, New York, United States.Show MeSH
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Mentions: We next asked whether phosphorylation of GST-CTD affects its stability in the proteasome assay. For this, we first used a recombinant c-Abl derivative to phosphorylate GST-CTD. This resulted in conversion of a fraction of the GST-CTD to a low-mobility, Tyr1-P isoform, although the majority remained unphosphorylated (Figure 3C, lane 1, Figure 3—figure supplement 1A), consistent with the processive phosphorylation by c-Abl observed previously (Duyster et al., 1995). We then performed the proteasome assay described above using c-Abl-phosphorylated GST-CTD (Figure 3C). Strikingly, the Tyr1 hyperphosphorylated GST-CTD (top panel, upper band, and lower panel) was resistant to degradation (lane 2), while the remaining unphosphorylated GST-CTD (top panel, bottom band) was degraded. Addition of 0.01% SDS again promoted degradation of unphosphorylated GST-CTD, but the Tyr1-P isoform remained resistant (lane 3). Significantly, GST-CTD phosphorylated by the Ser5/Ser7 kinase CDK7, which converted essentially all of the substrate to the hyperphosphorylated form, was not protected from degradation (lanes 4–6), indicating a specific role of Tyr1-P in preventing proteasomal degradation.
Affiliation: Department of Biological Sciences, Columbia University, New York, United States.