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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

ABSTRACT: To investigate the whole-transcriptome effects of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)/SCAF6 which specifically binds to the Ser2 of C-terminal domain of RNA Polymerase II, we established HEK293 cell line in which SCAF6 gene was knocked down by shRNA. We then performed gene expression and alternative splicing profiling using data obtained from RNA-seq of 2 cell lines (HEK293 Control vs HEK293 SCAF6 KD).

ORGANISM(S): Homo sapiens

PROVIDER: GSE221328 | GEO | 2023/08/17

REPOSITORIES: GEO

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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:We have discovered distinctive protein interactomes for Ser2 and Ser5 phosphorylation of the C-terminal domain (CTD) of RNA Pol II. Calcium Homeostasis Endoplasmic reticulum (CHERP) was identified in our pulldown to directly interact with Ser2 phosphomarks on the CTD of Pol II. As Pol II is a chromatin-associated protein, we sought to identify genomic regions that CHERP, a Pol II binding protein, occupy through ChIP-seq analysis.

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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modification on the C-terminal domain (CTD) of the largest subunit , generating a sophisticated PTM landscape for the spatiotemporal recruitment to transcriptional regulators. To delineate the protein interactomes recruited to Pol II at different stages of transcription, we in vitro reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 positions, the hallmark phosphorylation at the initation and productive elongation stages of transcription, respectively. Distinctive protein interactomes indicates different proteins are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) specifically binds to the Ser2 of the heptad. The loss of the interaction between CHERP and Pol II results in broad alternative splicing events. Our method points to a new method to distinguish the PTM codes that coordinate the transcription process.

2023-02-06 | MSV000091213 | MassIVE

Distinctive interactomes of RNA polymerase II phosphorylation during different stages of transcription.

Project description: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, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription.

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CHERP controls alternative mRNA splicing in human cell line

Project description:Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) is co-localized with inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or peri-nuclear part, and has been considered to have a role in intracellular calcium signaling. Recently, it has recognized that CHERP structurally carries nuclear localization signal and arginine/serine-dipeptide repeats like domain, and interacts with spliceosome. In this study, we showed that poly (A)+ RNA was accumulated in the nucleus on CHERP depletion. Using the global analysis, CHERP regulated alternative mRNA splicing events through the interaction with U2 snRNP and U2 snRNP related proteins. Our analysis indicated that intron retention was most frequently observed among five alternative splicing patterns in accordance with the accumulation of poly (A)+ RNA in the nucleus. Further, intron retention and cassette exon choice were influenced by the strength of 5’ or 3’ splice site, GC content, or intron length. In addition, CHERP depletion induced the abnormality of cell cycle progression at M phase and cell division. These results suggested that CHERP was involved in the regulation of alternative splicing.

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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

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Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

Project description:Distinctive Interactomes of RNA polymerase II phosphorylation during different stages of transcription

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TRIM28 regulates RNA polymerase II promoter proximal pausing and entry into processive elongation

Project description:Analysis of total PolII and Ser2-phosphorylated PolII genomic occupancy in control-transfected (WT) or Trim28-knockdown (Trim28-KD) embryonic stem cells. Embryonic stem cells were transfected with luciferase siRNA (WT) or Trim28 siRNA (Trim28 KD) in duplicate experiments. 48 hrs after transfection, cells were crosslinked with formaldehyde, collected, lysed, and chromatin was fragmented with sonication. Total PolII or Ser2-phosphorylated PolII genomic occupancy in WT or Trim28 KD cells were determined by ChIP-seq.

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2022-01-12 | PXD020367 | Pride

Generalized and social anxiety disorder interactomes show distinctive overlaps with striosome and matrix interactomes.

Project description:Mechanisms underlying anxiety disorders remain elusive despite the discovery of several associated genes. We constructed the protein-protein interaction networks (interactomes) of six anxiety disorders and noted enrichment for striatal expression among common genes in the interactomes. Five of these interactomes shared distinctive overlaps with the interactomes of genes that were differentially expressed in two striatal compartments (striosomes and matrix). Generalized anxiety disorder and social anxiety disorder interactomes showed exclusive and statistically significant overlaps with the striosome and matrix interactomes, respectively. Systematic gene expression analysis with the anxiety disorder interactomes constrained to contain only those genes that were shared with striatal compartment interactomes revealed a bifurcation among the disorders, which was influenced by the anterior cingulate cortex, nucleus accumbens, amygdala and hippocampus, and the dopaminergic signaling pathway. Our results indicate that the functionally distinct striatal pathways constituted by the striosome and the matrix may influence the etiological differentiation of various anxiety disorders.

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Ribosome footprinting in the cytosol and endoplasmic reticulum

Project description:In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a role for the ER in global protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression. HEK293 cells were fractionated between the cytosol and endoplasmic reticulum. Within each fraction, ribosome footprints were generated and sequenced. In parallel, total mRNA was sequenced.

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