Dataset Information

RNA polymerase III interactome

ABSTRACT: Many regulatory proteins and complexes have been identified which influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding RNAs. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used proteomic approaches and pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to find identify the potential interactors through mass spectrometry-based proteomics. A large number of proteins were found in the interactome, which includes known chromatin modifiers, factors and regulators of transcription by pol I and pol II.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Yohann Couté

LAB HEAD: Virginie Brun

PROVIDER: PXD003748 | Pride | 2019-01-02

REPOSITORIES: pride

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Publications

Interactome of the yeast RNA polymerase III transcription machinery constitutes several chromatin modifiers and regulators of the genes transcribed by RNA polymerase II.

Bhalla Pratibha P Vernekar Dipti Vinayak DV Gilquin Benoit B Couté Yohann Y Bhargava Purnima P

Gene 20181226

Eukaryotic transcription is a highly regulated fundamental life process. A large number of regulatory proteins and complexes, many of them with sequence-specific DNA-binding activity are known to influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes genes encoding small, stable, non-translated RNAs. The pol III transcription is precisely regulated under various stress conditions. We used po ...[more]

PMID: 30593915

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Project description:RNA polymerase (RNA Pol) III synthesizes the tRNAs, the 5S ribosomal RNA and a small number of untranslated RNAs. In vitro, it also transcribes short interspersed nuclear elements (SINEs). We investigated the distribution of RNA Pol III and its associated transcription factors on the genome of mouse embryonic stem (ES) cell using a highly specific tandem ChIP-Seq method. Only a subset of the annotated class-III genes was bound and thus transcribed. A few hundred SINEs were associated with the RNA Pol III transcription machinery. We observed that RNA Pol III and its transcription factors were present at thirty unannotated sites on the mouse genome, only one of which was conserved in human. An RNA was associated with more than 80% of these regions. More than 2200 regions bound by TFIIIC transcription factor were devoid of RNA Pol III. These sites are correlated with association of CTCF and the cohesin. Cohesin has been shown to occupy sites bound by CTCF and to contribute to DNA loop formation associated with gene repression or activation. This observation suggests that TFIIIC may play a role in chromosome organization in mouse. We also investigated the genome-wide distribution of the ubiquitous TFIIS variant, TCEA1. We found that, as in Saccharomyces cerevisiae, TFIIS is associated with class III genes and also with SINEs suggesting that TFIIS is a RNA Pol III transcription factor in mammals. We performed ChIP-seq experiment on mouse ES cells, in order to analyse the distribution of the RNA Pol III, with two of its subunits, RPC1 and RPC4, of the two distinct forms of the transcription factor TFIIIB, with BRF1 and BRF2, respectively subunit of TFIIIB-beta, and TFIIIB-alpha form, and three subunits of the transcription factor TFIIIC, TFIIIC90, TFIIIC110, TFIIIC220. We also analysed the distribution of the RNA Pol II elongation factor TCEA1. We used tagged proteins, in order to develop a highly specific and generic ChIP-seq protocol. A sequence encoding a 6 histidine-Flag-HA tag was inserted just after the last codon of the gene encoding proteins of the RNA Pol III machinery subunits, or just after the start codon for TCEA1, using the recombineering technology. Untagged ES cell line was used as negative control for data processing. Our dataset comprises of ten ChIP-seq samples, eight from tagged proteins, two from untagged cell line.

			Action	DRS
	Brf1-Act-R1.raw	Raw
	Brf1-Act-R2.raw	Raw
	Brf1-Rep-R1.raw	Raw
	Brf1-Rep-R2.raw	Raw
	CtrlBrf1-Act.raw	Raw