Targeted protein degradation reveals Pol II heterogeneity and functional diversity

Published: 8 August 2022| Version 1 | DOI: 10.17632/swyhf77565.1
Contributor:
yuanjun li

Description

Pol II subunits are considered to being involved in various transcription-associated processes. It is unclear whether they play differential regulatory roles in gene expression. Here, we performed nascent and mature transcript sequencing after acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription and carry out preferential regulation of RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to depletion of different Pol II subunits tend to be involved in diverse biological functions and have various RNA half-life. The sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of RNA polymerase II and different genes appear to depend on some of the subunits. The panels in Figure1B shows the western blot analyses of the protein levels of 12 Pol II subunits after IAA treatment at the indicated time in mESCs. The panels in Figure1D shows the western blot analyses of whole-cell extracts from each RPB degron cells treated with IAA at indicated time points. The panels in Figure2G shows the western blot analyses of indicated aliquots of fractions from the mESC chromatin extracts isolated by size exclusion chromatography on a Superdex 200 increase column. The panels in FigureS1A were the images of PCR-based genotyping of degron mESCs for each Pol II subunit, with the wild-type cell as the control. The panels in FigureS1E shows the western blot analyses of the expression of each subunit after 3 h of IAA treatment using antibodies against endogenous RPB subunits. The panels in FigureS1F shows the western blot analyses of efficient subunit degradation in RPB4, RPB7, RPB11 and RPB12 degron cells exposed to IAA for 96 h, as some cells remained alive. The panels in FigureS1G shows the western blot analyses of whole-cell extracts from Tir1 parental cells treated with IAA at indicated time points. The panels in FigureS6B shows the Immunofluorescence analyses of the distribution of the RPB1 signal (red) before and after the degradation of each Pol II subunit for 3 h. A decrease in the GFP signal indicates efficient degradation. DAPI staining indicates the nucleus. The panels in FigureS6C shows the immunofluorescence analyses of the distribution of the RPB1 signal (red) after degradation of the RPB3/RPB9/RPB10/RPB11 at the indicated time points.

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Pol II subunits are considered to being involved in various transcription-associated processes. It is unclear whether they play differential regulatory roles in gene expression. Here, we performed nascent and mature transcript sequencing after acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription and carry out preferential regulation of RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to depletion of different Pol II subunits tend to be involved in diverse biological functions and have various RNA half-life. The sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of RNA polymerase II and different genes appear to depend on some of the subunits. The panels in Figure1B shows the western blot analyses of the protein levels of 12 Pol II subunits after IAA treatment at the indicated time in mESCs. The panels in Figure1D shows the western blot analyses of whole-cell extracts from each RPB degron cells treated with IAA at indicated time points. The panels in Figure2G shows the western blot analyses of indicated aliquots of fractions from the mESC chromatin extracts isolated by size exclusion chromatography on a Superdex 200 increase column. The panels in FigureS1A were the images of PCR-based genotyping of degron mESCs for each Pol II subunit, with the wild-type cell as the control. The panels in FigureS1E shows the western blot analyses of the expression of each subunit after 3 h of IAA treatment using antibodies against endogenous RPB subunits. The panels in FigureS1F shows the western blot analyses of efficient subunit degradation in RPB4, RPB7, RPB11 and RPB12 degron cells exposed to IAA for 96 h, as some cells remained alive. The panels in FigureS1G shows the western blot analyses of whole-cell extracts from Tir1 parental cells treated with IAA at indicated time points. The panels in FigureS6B shows the Immunofluorescence analyses of the distribution of the RPB1 signal (red) before and after the degradation of each Pol II subunit for 3 h. A decrease in the GFP signal indicates efficient degradation. DAPI staining indicates the nucleus. The panels in FigureS6C shows the immunofluorescence analyses of the distribution of the RPB1 signal (red) after degradation of the RPB3/RPB9/RPB10/RPB11 at the indicated time points.

Institutions

Peking University

Categories

Protein Degradation, RNA Polymerase

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