What's all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain<i>via</i>mass spectrometry

LeBlanc, Blase M.; Moreno, Rosamaria Y.; Escobar, Edwin E.; Ramani, Mukesh Kumar Venkat; Brodbelt, Jennifer S.; Zhang, Yan Jessie

doi:10.1039/d1cb00083g

Cited by 5 publications

(3 citation statements)

References 129 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In conclusion, many techniques are now available to study the CTD code�mass spectrometry in particular 71 �but chemical synthesis, coupled with biological assays (ELISA, FP, and others), will be central to further discoveries due to their unrivaled precision. The CTD code is complicated and still not fully understood, but knowing more about the limitations of the main tools for detecting and mapping phosphorylations provides powerful insight in the quest to crack the code.…”

Section: ■ Conclusionmentioning

confidence: 99%

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis

Piemontese,

Herfort,

Perevedentseva

et al. 2024

J. Am. Chem. Soc.

View full text Add to dashboard Cite

Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48−90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.

show abstract

Section: ■ Conclusionmentioning

confidence: 99%

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis

Piemontese,

Herfort,

Perevedentseva

et al. 2024

J. Am. Chem. Soc.

View full text Add to dashboard Cite

show abstract

“…It is worth mentioning that the antibody used in Figure 2 recognizes all RPB1 CTD forms, however, a plethora of antibodies characteristic for specific stages of transcription exists. Human RPB1 CTD contains 52 heptad repeats that can be reversibly phosphorylated and the specific phosphorylation pattern reflects subsequent steps of transcription [103][104][105], Importantly, distinct steps of transcription could be therapeutically targeted in cancer and other complex diseases [106]. Immunolabeling with antibodies against distinct RPB1 CTD phosphorylation results in distinct spatio-temporal localization of RPB1 and reflects distinct stages of transcription.…”

Section: Dynamics Of Rnapii By Smlmmentioning

confidence: 99%

How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

Hoboth

Šebesta

Hozák

2021

IJMS

View full text Add to dashboard Cite

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

show abstract

“…Determining the function of the phospho‐CTD marks relies primarily on detection by specific antibodies (Eick & Geyer, 2013), mutagenesis (Hsin et al, 2011; Schwer & Schuman, 2011; Hintermair et al, 2012; Corden, 2013; Rosonina et al, 2014), chemical perturbation of transcriptional kinases (Akhtar et al, 2009; Kanin et al, 2007; Larochelle et al, 2012; Liu et al, 2004; Rodrίguez‐Molina et al, 2016; Tietjen et al, 2010), and mass spectrometry (Harlen et al, 2016; LeBlanc et al, 2021; Mayfield et al, 2017; Nemec et al, 2017; Schüller et al, 2016; Suh et al, 2016). While each approach has provided valuable insight into CTD specific functions, none are without bias.…”

Section: Introductionmentioning

confidence: 99%

The Phantom Mark: Enigmatic roles of phospho‐Threonine 4 modification of the C‐terminal domain of RNA polymerase II

2023

View full text Add to dashboard Cite

The largest subunit of RNA polymerase II (Pol II) has an unusual carboxylterminal domain (CTD). This domain is composed of a tandemly repeating heptapeptide, Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , that has multiple roles in regulating Pol II function and processing newly synthesized RNA. Transient phosphorylation of Ser2 and Ser5 of the YS 2 PTS 5 PS repeat have well-defined roles in recruiting different protein complexes and coordinating sequential steps in gene transcription. As such, these phospho-marks encipher a molecular recognition code, colloquially termed the CTD code. In contrast, the contribution of phospho-Threonine 4 (pThr4/pT4) to the CTD code remains opaque and contentious. Fuelling the debate on the relevance of this mark to gene expression are the findings that replacing Thr4 with a valine or alanine has varied impact on cellular function in different species and independent proteomic analyses disagree on the relative abundance of pThr4 marks. Yet, substitution with negatively charged residues is lethal and even benign mutations selectively disrupt synthesis and 3 0 processing of distinct sets of coding and non-coding transcripts. Suggestive of non-canonical roles, pThr4 marked Pol II regulates distinct gene classes in a species-and signal-responsive manner. Hinting at undiscovered roles of this elusive mark, multiple signal-responsive kinases phosphorylate Thr4 at target genes. Here, we focus on this under-explored residue and postulate that the pThr4 mark is superimposed on the canonical CTD code to selectively regulate expression of targeted genes without perturbing genome-wide transcriptional processes.

show abstract

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

Abstract: RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

Cited by 5 publications

References 129 publications

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis

Multiphosphorylation-Dependent Recognition of Anti-pS2 Antibodies against RNA Polymerase II C-Terminal Domain Revealed by Chemical Synthesis

How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

The Phantom Mark: Enigmatic roles of phospho‐Threonine 4 modification of the C‐terminal domain of RNA polymerase II

Contact Info

Product

Resources

About