Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification

Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

doi:10.1016/j.gene.2014.07.070

Cited by 11 publications

(10 citation statements)

References 44 publications

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“…1A). A large number and variety of associated protein complexes were detected in this study, which include in the active state, 30 out of total 44 interactors of Rpc160, reported previously (50). Venn intersections of the interactome of the individual baits found a comparatively smaller number of proteins interacting with all three of them under both the states ( Fig.…”

Section: Many Interactors Of Pol III Tc Are Common Between Active Andsupporting

confidence: 71%

Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

Bhalla

Vernekar

Shukla

et al. 2018

Preprint

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Many regulatory proteins and complexes influence transcription by RNA polymerase (pol) II.In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding genes. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to identify potential interactors through mass spectrometry-based proteomics. A large interactome constituting known chromatin modifiers, factors and regulators of transcription by pol I and pol II revealed the possibility of a large number of signaling cues for pol III transcription against adverse conditions. We found one of the pol II-associated factors, Paf1 complex (PAF1C) interacts with the three baits. Its occupancy on the pol III-transcribed genes is low and not correlated with pol III occupancy. Paf1 deletion leads to higher occupancy of pol III, γ-H2A and DNA pol2 but no change in nucleosome positions. Genotoxins exposure causes pol III but not Paf1 loss from the genes. PAF1C promotes the pol III pausing and restricts its accumulation on the genes, which reduces the replication stress caused by the pol III barrier and transcriptionreplication conflict on these highly transcribed genes.

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Section: Many Interactors Of Pol III Tc Are Common Between Active Andsupporting

confidence: 71%

Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

Bhalla

Vernekar

Shukla

et al. 2018

Preprint

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“…However, we suggest that Bdp1 could induce the displacement of the τB module as a regulatory mechanism essential for the initial round of Pol III transcription. Consistent with this model, a recent mass spectrometry study of TAP-purified Pol III from actively transcribing yeast cells detected co-eluting peptides from all three subunits of TFIIIB and peptides from the τ131 subunit, but not from any τB subunit 39 .…”

Section: Discussionmentioning

confidence: 55%

Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly

et al. 2015

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In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131–τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.

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“…To identify yeast proteins associated with Ty1 IN that could regulate Ty1 replication, we adapted the tandem chromatin affinity purification procedure after in vivo cross-link (TChAP), which we developed previously (41) (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

“…IN protein was expressed with a C-terminal histidine-biotin-histidine (HBH) tag (42), allowing ectopic IN-HBH purification under denaturing conditions. Proteins that co-purified with IN-HBH were compared to those detected in the control yeast strain expressing untagged IN, and IN-HBH specific partners were sorted according to the settings of TChAP selection criteria (41). Ultimately, we identified a set of 265 IN binding proteins (Table S1), which expands the number of candidates previously described (37).…”

Section: Identification Of Ty1 Integrase Binding Partnersmentioning

confidence: 99%

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

Barkova

Adhya

Conesa

et al. 2022

Preprint

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Background Transposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN). Results Here, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. Conclusion Our study highlights an important role of CK2 in the regulation of Ty1 retrotransposition. We provide the first evidence that Ty1 IN is post-translationally modified in vivo, as observed for retroviral INs, and demonstrate that CK2 strongly represses Ty1 mobility by inhibiting Ty1 transcription. The proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

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Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification

Cited by 11 publications

References 44 publications

Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition

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