Mapping RNAPII CTD Phosphorylation Reveals That the Identity and Modification of Seventh Heptad Residues Direct Tyr1 Phosphorylation

Abstract

The C-terminal domain (CTD) of the largest subunit in eukaryotic RNA polymerase II has a repetitive heptad sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 which is responsible for recruiting transcriptional regulatory factors. The seventh heptad residues in mammals are less conserved and subject to various post-translational modifications, but the consequences of such variations are not well understood. In this study, we use ultraviolet photodissociation mass spectrometry, kinetic assays, and structural analyses to dissect how different residues or modifications at the seventh heptad position alter Tyr1 phosphorylation. We found that negatively charged residues in this position promote phosphorylation of adjacent Tyr1 sites, whereas positively charged residues discriminate against it. Modifications that alter the charges on seventh heptad residues such as arginine citrullination negate such distinctions. Such specificity can be explained by conserved, positively charged pockets near the active sites of ABL1 and its homologues. Our results reveal a novel mechanism for variations or modifications in the seventh heptad position directing subsequent phosphorylation of other CTD sites, which can contribute to the formation of various modification combinations that likely impact transcriptional regulation.

Evaluating Spatiotemporal Dynamics of Phosphorylation of RNA Polymerase II Carboxy-Terminal Domain by Ultraviolet Photodissociation Mass Spectrometry

“…UVPD has been used for the qualitative characterization of phosphopeptides in several recent studies aimed at elucidating sites of CTD phosphorylation of RNAP II. − ,− Here, we extend the use of 193 nm UVPD to a quantitative PRM workflow (Figure ). This study establishes the capacity of UVPD-based PRM analysis for determining with spatiotemporal resolution the dynamic site-specific pattern of phosphorylation of the CTD of RNA polymerases II by kinases.…”

“…Fragments were matched to the nine ion types observed from UVPD of peptides ( a , a +1, b , c , x , x +1, y , y –1, z ) using ProSight Lite. As reported before, phosphosite confirmation was achieved by adding the mass of a phospho group (+79.97 Da) at each of the possible Tyr or Ser residues to identify fragment ions that were phosphorylated. ,,, All PRM-MS raw files were processed in Skyline v20.1 to generate XICs and allow targeted peak integration . The summed peak areas of the 3 to 5 most abundant fragment ions that allowed confident phosphosite differentiation of peptides were used to quantify CTD phosphopeptides.…”

“…Alternate MS/MS methods offer new opportunities for enhancing the successful differentiation of isoform-specific peptides, particularly those having the same sequence but different sites of modification. Ultraviolet photodissociation (UVPD) is one of the newer ion activation methods that has emerged for proteomics applications. , In addition to promoting extensive backbone cleavages of peptides that result in high sequence coverage, UVPD preserves labile post-translational modifications, allowing confident localization of PTMs. − These features make UVPD a compelling option for implementation in quantitative proteomic workflows such as PRM. In this work, we report a quantitative method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the periodic CTD sequence by the cyclin-dependent kinase 7 (CDK7) kinase module using UVPD for peptide identification and phosphosite localization.…”

What's all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domainviamass spectrometry

“…The major phosphorylation product appears in the LC trace during MS analysis as a CTD peptide with phosphoryl tyrosine flanking a negative residue (such as phosphoryl Ser7). 58 Such a preference leads to the routine generation of doubly phosphorylated Ser7Tyr1. Thus, the intelligent use of MS has proven critical in delineating the crosstalk between different phosphorylation sites of the CTD.…”

Characterization of HLA-A*02:01 MHC Immunopeptide Antigens Enhanced by Ultraviolet Photodissociation Mass Spectrometry

“…UVPD has garnered growing attention as an alternative fragmentation method for peptides and proteins. − Two hallmarks of UVPD are the generation of a wide variety of fragment ions, including but not limited to a , b , c , d , x , y , z , and w ions, and the retention of PTMs. − The variety of fragment ions typically leads to a higher sequence coverage. Another feature of UVPD is the lack of significant charge state dependence compared to other fragmentation techniques. , UVPD has been shown to generate both d and w ions from side chain losses, each affording diagnostic neutral losses, which facilitate differentiation of leucine and isoleucine. , Moreover, the production of both d and w ions by UVPD offers the opportunity for bidirectional cleavages that can double the number of potential fragment ions, augmenting successful leucine and isoleucine differentiation.…”