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 6 publications

(4 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

“…Relative quantitative proteomics has been instrumental in driving discovery-based explorations into the dynamic expression of proteins and their modifications across various biological and disease states. In recent years, it has played a pivotal role in elucidating insights across diverse domains, ranging from the pathobiology of SARS-CoV2 viral infection [1][2][3] to the molecular mechanisms underlying RNA polymerase II transcription machinery [4][5][6] . Commonly used techniques in the realm of relative quantitative proteomics, performed without prior knowledge of the peptide or protein composition of the sample, include Label-Free Quantification (LFQ), which, as the name implies, enables relative quantitation without relying on any chemical labels.…”

Section: Introductionmentioning

confidence: 99%

MAGMa: Your Comprehensive Tool for Differential Expression Analysis in Mass-Spectrometry Proteomic Data

Gupta,

Kang,

Sun

et al. 2024

Preprint

View full text Add to dashboard Cite

Proteomics, the study of proteins and their functions, plays a vital role in understanding biological processes. In this study, we sought to address the challenges in analyzing complex proteomic datasets, where subtle changes in protein abundance are difficult to detect. Utilizing a newly developed tool,MaximalAggregation ofGood protein signal fromMass spectrometric data (MAGMa), we demonstrated its superior performance in accurately identifying true signals while effectively filtering out noise. Here we show that MAGMa strikes a balance between sensitivity and specificity on benchmarking datasets, offering a robust solution for analyzing various quantitative proteomic datasets. These findings advance the field by providing researchers with a powerful tool to uncover subtle changes in protein abundance, contributing to our understanding of complex biological systems and potentially facilitating the discovery of new therapeutic targets.

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 6 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

MAGMa: Your Comprehensive Tool for Differential Expression Analysis in Mass-Spectrometry Proteomic Data

Contact Info

Product

Resources

About