Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

Abstract

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

A combinatorial view of old and new RNA polymerase II modifications

“…However, in mammals, Tyr phosphorylation occurs primarily at the 5ʹ end of genes [80,163]. A recent study showed that Tyr1 phosphorylation by c-Abl selectively directs P-TEFb/ CDK9 to phosphorylate Ser2 [108] (Figure 4c). In biochemical studies, CDK9 can phosphorylate both Ser2 and Ser5 [107], but in vivo it specifically targets Ser2 [105].…”

“…P-TEFb is composed of a regulatory cyclin and catalytic CDK9 component, which phosphorylates Ser2 residues to enhance polymerase processivity [102][103][104]106]. The ability of P-TEFb to phosphorylate Ser2 is primed by previous phosphorylation events at Ser7 [107] and Tyr1 [108]. A recent study in a breast cancer cell line identified citrullination of Arg1810 by peptidyl arginine deiminase 2 (PADI2), a calcium-dependent enzyme, as a key CTD modification for recruitment of P-TEFb to the paused polymerase of genes involved in cell proliferation [109].…”

Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates

“…This work is supported by grants from the National Institutes of Health (R01 GM104896 and 125882 to YJZ) and Welch Foundation (F-1778 to YJZ). Methods presented here are adapted from previous work (Mayfield et al, 2019).…”

“…Distinct pools of unphosphorylated and hyperphosphorylated RPB1 are detected in cell lysates using polyacrylamide gel electrophoresis (PAGE) due to a dramatic shift in the isoelectric point of RPB1 resulting from hyperphosphorylation. This physical characteristic of hyperphosphorylated RPB1 allowed for the initial discovery and characterization of the CTD and continues to be a useful tool in the study of CTD phosphorylation (Corden et al, 1985;Mayfield et al, 2019).…”

“…Subclone GST-yeast CTD sequence (as described inMayfield et al, 2019) into the pET-28 a (+) vector, or equivalent, using any standard cloning method.2. Transform sequence-verified plasmid into BL21 (DE3) Competent E. coli cells using conventional methods and select for transformants by growing overnight at 37 °C on an agar plate with 50 μg/ml kanamycin.3.…”