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Proteomic study of pCTD interactomes (A) In vitro phosphorylation of a GST-CTD recombinant protein containing 26x repeat consensus CTD heptads is treated with TFIIH or DYRK1A and incubated with lysate from HEK-293 cells. A no-kinase sample is treated parallelly as a control. Affinity chromatography immobilizes the GST-tagged substrate and pulls down Pol II-interacting proteins. LC-MS/MS analysis identifies proteins in each sample. (B) Kinase activity assay of wild-type yCTD by TFIIH (dark blue) and DYRK1A (purple) fitted to the Michaelis-Menten kinetic equation. The Michaelis-Menten kinetic parameters k cat /K m (mM À1 min À1 ) are given for each respective fit. Each measurement was conducted in triplicate, with standard deviations shown as error bars. (C) Volcano plots comparing the pSer5 IP and the pSer2 IP (D). Both use unphosphorylated CTD IP as control. Enriched factors were determined using a p value of 0.05 and shown as dark gray dots. Factors mentioned in the text are labeled and shown as red dots. (E) Gene ontology terms enriched for the top 100 proteins identified in the phospho-CTD interactome data for pSer2. Visualization was done with ShinyGO 0.76.
Source publication
During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, re...
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... a thorough bioinformatic and structural search of CTD kinases, we identified DYRK1A as a candidate due to its signature motif that supports Ser2 phosphorylation. 21 Subsequent enzymatic reactions with four heptad repeats resulted in products exclusively phosphorylated at the Ser2 in the context of the consensus sequence of YSPTSPS ( Figures 1D and S3). Lack of phosphorylation on the fourth repeat by DYRK1A is likely a result of higher entropy cost at the end of the flexible peptide. ...
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... conducted label-free proteomics analyses of pulldowns using phosphorylated CTDs that were differentially treated by kinases with well-characterized specificity ( Figures 3A, and S8A). The bait used in the proteomic study was a GST-tagged 26-repeat CTD, consisting mostly of a consensus sequence, which was treated with different kinases (TFIIH kinase module and Dyrk1a). ...
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... bait used in the proteomic study was a GST-tagged 26-repeat CTD, consisting mostly of a consensus sequence, which was treated with different kinases (TFIIH kinase module and Dyrk1a). The 26-repeat CTD is a good substrate for both kinases, and the kinetic experiments revealed that this recombinant CTD was phosphorylated effectively by TFIIH and Dyrk1a with Figure 3B). The activity of TFIIH is much higher on a longer repeat than on a CTD substrate with only four repeats ( Figure 1B). ...
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... the samples were analyzed using label-free proteomics by comparing the abundance of pulleddown proteins in each kinase-treated sample to that in the control. Different from the previous efforts in obtaining differential phosphorylated CTD, our study's interactomes of pSer2 and pSer5 are dramatically different, identifying several dozen proteins with differential binding patterns ( Figures 3C and 3D). For pSer5 pulldown, a significant characteristic was the reduction of many proteins upon Ser5 phosphorylation compared to the control ( Figure 3C). ...
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... from the previous efforts in obtaining differential phosphorylated CTD, our study's interactomes of pSer2 and pSer5 are dramatically different, identifying several dozen proteins with differential binding patterns ( Figures 3C and 3D). For pSer5 pulldown, a significant characteristic was the reduction of many proteins upon Ser5 phosphorylation compared to the control ( Figure 3C). Many of these proteins were histones, histone variants, and associated accessory proteins, such as chromatin remodeling complexes ( Figure 3C; and Table S1). ...
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... pSer5 pulldown, a significant characteristic was the reduction of many proteins upon Ser5 phosphorylation compared to the control ( Figure 3C). Many of these proteins were histones, histone variants, and associated accessory proteins, such as chromatin remodeling complexes ( Figure 3C; and Table S1). This observation is consistent with the biological understanding that active transcription reduces chromatin density by ejecting and relocating them. ...
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... observation is consistent with the biological understanding that active transcription reduces chromatin density by ejecting and relocating them. 25 A top hit for depletion upon Ser5 phosphorylation is Heterochromatin protein 1-binding protein 3 (HP1B3), a component that maintains heterochromatin condensation ( Figure 3C). Other top hits include AIP E3 ligase homologs, which have been implicated in RPB1 translocation and degradation. ...
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... most significant hit for pSer5 binding was mRNA capping enzyme Cap1 2 0 O-ribose methyltransferase 1, a major function of Ser5 phosphorylation. 27,28 Furthermore, PHF3, a recently identified CTD-binding protein, was highly enriched, consistent with recent reports that it can directly bind Ser5 and/or Ser2 phosphorylated CTD 29 ( Figure 3C). ...
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... pattern of pSer2 pulldown ( Figure 3D) showed a significant difference from that of pSer5 pulldown ( Figure 3C), with more proteins associated with the phosphoryl mark rather than depletion. Some previously characterized pSer2-binders, such as PHF3, PCF11, 30 PHRF1, 31 and RPRD2 32 are among the hits, validating the accuracy of our pulldown ( Figure 3D). ...
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... pattern of pSer2 pulldown ( Figure 3D) showed a significant difference from that of pSer5 pulldown ( Figure 3C), with more proteins associated with the phosphoryl mark rather than depletion. Some previously characterized pSer2-binders, such as PHF3, PCF11, 30 PHRF1, 31 and RPRD2 32 are among the hits, validating the accuracy of our pulldown ( Figure 3D). ...
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... pattern of pSer2 pulldown ( Figure 3D) showed a significant difference from that of pSer5 pulldown ( Figure 3C), with more proteins associated with the phosphoryl mark rather than depletion. Some previously characterized pSer2-binders, such as PHF3, PCF11, 30 PHRF1, 31 and RPRD2 32 are among the hits, validating the accuracy of our pulldown ( Figure 3D). Many proteins identified are spliceosome components such as U5 snRNP and splicing factors, but their direct interaction with CTD has yet to be established. ...
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... large fraction of the bound proteins are implicated in association with RNA. Ontology analysis of the top 100 enriched hits from the pSer2 pulldown revealed that RNA-binding proteins were the most enriched category ( Figure 3E). Most proteins identified involve mRNA processing, translocation, or post-transcriptional modifications. ...
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... is highly enriched in both pSer2 and pSer5 interactomes. We identified two PP1 catalytic subunit isoforms, alpha and gamma, and TOX high-mobility group box family member 4, which forms a stable complex with PNUTS/PP1 phosphatase complex ( Figures 3C and 3D). The PNUTS/PP1 phosphatase complex is known to be involved in Pol II dephosphorylation during active transcription. ...
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Citations
The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3′ end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.
Decades of scientific research have been devoted to unraveling the intricacies of eukaryotic transcription since the groundbreaking discovery of eukaryotic RNA polymerases in the late 1960s. RNA polymerase II, the polymerase responsible for mRNA synthesis, has always attracted the most attention. Despite its structural resemblance to its bacterial counterpart, eukaryotic RNA polymerase II faces a unique challenge in progressing transcription due to the presence of nucleosomes that package DNA in the nuclei. In this review, we delve into the impact of RNA polymerase II and histone signaling on the progression of eukaryotic transcription. We explore the pivotal points of interactions that bridge the RNA polymerase II and histone signaling systems. Finally, we present an analysis of recent cryo-electron microscopy structures, which captured RNA polymerase II-nucleosome complexes at different stages of the transcription cycle. The combination of the signaling crosstalk and the direct visualization of RNA polymerase II-nucleosome complexes provides a deeper understanding of the communication between these two major players in eukaryotic transcription.
