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R2TP_BioID-MS - R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

ABSTRACT: The R2TP/Prefoldin-like (R2TP/PFDL) complex is a cochaperone complex involved in the assembly of a number of critical protein complexes. Here we use proximity-dependent biotinylation (BioID) coupled to mass spectrometry to confirm interactions of ZNHIT2, a cofactor of R2TP/PFDN, identified by TAP-MS.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Christian Poitras

LAB HEAD: Benoit Coulombe

PROVIDER: PXD006200 | Pride | 2018-10-24

REPOSITORIES: Pride

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	BioID-files.xlsx	Xlsx
	F012170.dat	Other
	VL_20170109_COU_02.raw	Raw

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Publications

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein.

Cloutier Philippe P Poitras Christian C Durand Mathieu M Hekmat Omid O Fiola-Masson Émilie É Bouchard Annie A Faubert Denis D Chabot Benoit B Coulombe Benoit B

Nature communications 20170531

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the ...[more]

PMID: 28561026

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R2TP_TAP-MS - R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

Project description:The R2TP/Prefoldin-like (R2TP/PFDL) complex is a cochaperone complex involved in the assembly of a number of critical protein complexes. Here we use multiple target tandem affinity purification (TAP) coupled to mass spectrometry to map out the interaction network around R2TP/PFDL.

2018-10-24 | PXD006198 | Pride

R2TP_SILAC_FLAG-MS - R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

Project description:The R2TP/Prefoldin-like (R2TP/PFDL) complex is a cochaperone complex involved in the assembly of a number of critical protein complexes. Here we use stable isotope labeling with amino acids in cell culture (SILAC) and FLAG tag affinity purification coupled to mass spectrometry to assess whether siRNA-driven knockdown of cofactors or subunits of R2TP/PFDN affects composition of U5 snRNP and TSC complex.

2017-11-02 | PXD006199 | Pride

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein.

Project description:The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.

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RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

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In vivo and in vitro characterization and structural analysis of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases

Project description:DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigations involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA+ family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built 3D structural models of DPCD alone and in complex with R1R2 validated by experimental data combining Small-Angle X-ray Scattering and Cross-linking Mass Spectrometry, among others. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

2023-04-06 | PXD031339 | Pride

Identification of RUVBL1 and RUVBL2 as Novel Cellular Interactors of the Ebola Virus Nucleoprotein.

Project description:Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.

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RUVBL1/RUVBL2 ATPase Activity Drives PAQosome Maturation, DNA Replication and Radioresistance in Lung Cancer.

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CryoEM of RUVBL1-RUVBL2-ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8.

Project description:Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1-RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1-RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP.

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