SILAC APMS analysis of SRP14 interactome in HEK-293 cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF-X
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hek-293 Cell
SUBMITTER: 
Franck Vandermoere
LAB HEAD: Dr Severine Massenet
PROVIDER: PXD051488 | Pride | 2024-06-07
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 10 |
Similar Datasets
Project description:The Signal Recognition Particle (SRP) is a universally conserved and abundant ribonucleoprotein particle (RNP) in cells. Its composition and size vary across evolution. In mammals, SRP comprises one RNA molecule, the 7SL RNA, and six proteins: SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72. SRP is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. To reveal novel aspects of SRP biogenesis, we established the interactome of the GFP-SRP72 protein. To do so, we have produced stable human U2OS cell line expressing GFP-SRP72 and performed stable isotope labeling by amino acids in cell culture (SILAC) proteomic experiment using GFP-SRP72 as bait.
2024-06-07 | PXD042191 | Pride
Project description:The Signal Recognition Particle (SRP) is a universally conserved and abundant ribonucleoprotein particle (RNP) in cells. Its composition and size vary across evolution. In mammals, SRP comprises one RNA molecule, the 7SL RNA, and six proteins: SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72. SRP is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. To reveal novel aspects of SRP biogenesis, we established the interactome of the GFP-SRP72 protein. To do so, we have produced stable human U2OS cell line expressing GFP-SRP9 and performed stable isotope labeling by amino acids in cell culture (SILAC) proteomic experiment using GFP-SRP9 as bait.
2024-06-07 | PXD042192 | Pride
Project description:The Signal Recognition Particle (SRP) is a universally conserved and abundant ribonucleoprotein particle (RNP) in cells. Its composition and size vary across evolution. In mammals, SRP comprises one RNA molecule, the 7SL RNA, and six proteins: SRP19, SRP19, SRP19, SRP54, SRP68, and SRP72. SRP is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. To reveal novel aspects of SRP biogenesis, we established the interactome of the GFP-SRP72 protein. To do so, we have produced stable human U2OS cell line expressing GFP-SRP19 and performed stable isotope labeling by amino acids in cell culture (SILAC) proteomic experiment using GFP-SRP19 as bait.
2024-06-07 | PXD042195 | Pride
Project description:The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. We performed extensive analysis of GAR1, NHP2 and NAF1 proteomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, suggesting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases.
2022-02-17 | PXD026747 | Pride
Project description:The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. We performed extensive analysis of GAR1, NHP2 and NAF1 proteomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, suggesting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases.
2023-05-10 | PXD035330 | Pride
Project description:The conserved box H/ACA RNPs consist of one box H/ACA RNA and 4 core proteins: Dyskerin, NHP2, NOP10 and GAR1. The assembly of the H/ACA RNPs is a stepwise process which requires several assembly factors: SHQ1, NAF1, the Survival of Motor Neuron complex SMN, and the R2TP complex. It implicates the co-transcriptional assembly of a pre-particle containing the nascent RNAs, Dyskerin, NOP10, NHP2 and NAF1. The latest keeps the H/ACA RNP inactive, and needs to be replaced by GAR1 to produce mature and functional H/ACA RNPs in the Cajal bodies. In this study, we explore in details the molecular mechanism leading to the assembly of mature box H/ACA RNPs. We performed extensive analysis of GAR1, NHP21 and NAF interactomes by quantitative stable isotope labeling by amino acids in cell culture proteomic analyses (SILAC), and revealed new assembly intermediates: 1) an early protein-only pre-H/ACA RNP complex containing the core proteins DKC1, NOP10 and NHP2, and the assembly factors SHQ1 and NAF1, and 2) a late assembly intermediates containing the RNA, the core proteins and NAF1. We showed that the SMN complex is associated with late forming H/ACA pre-RNP complexes containing GAR1. Moreover, our study identifies new proteins highly and specifically associated with GAR1, NHP2 and NAF1, reflecting their importance in box H/ACA assembly or function, such as the PRMT1 and PRMT5 arginine methylases. MS analysis of purified GAR1 revealed new sites of arginine methylations in the two GAR domains of GAR1, and showed that most of these methylated arginines exist both in mono-methylated and di-methylated forms in cells. By different methods, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs. Therefore, GAR1 methylation should be important for its function.
2023-05-10 | PXD026749 | Pride
Project description:HSP90 and its co-chaperon R2TP are involved to assemble and stabilize many macro-molecular complexes involved in cell proliferation. R2TP complex is composed of RUVBL1 and RUVBL2 AAA+ ATPases and two adapter proteins RPAP3 and PIH1D1. The N-terminal domain of PIH1D1 was described to possess a basic pocket that binds phosphorylated peptides on some of the R2TP clients. Subunits of TSC complex (Tuberous SClerosis) have previously been described in proteomic analysis of proteins immune-precipitated with PIH1D1 N-terminal domain. TSC complex is a regulator of mTOR activity. It is composed of TSC1, TSC2 and TBC1D7 subunits. Here, we show that (i) TSC1 and TSC2 are both substrates of HSP90, (ii) the HSP90/R2TP system has multiple interaction with TSC complex subunits and (iii) it is functionally involved in the assembly of the TSC complex. We found a direct interaction of TSC1 with the phospho-binding pocket of PIH1D1 N-terminal domain regardless of phosphorylationbut in a non-canonical phosphorylation independent manner[XM1] . TSC2 and TBC1D7 would also interact with R2TP but independently of PIH1D1. Taken together, these data suggest that R2TP acts as a platform for the independent recruitment of TSC subunits before the assembly of the TSC complex
2024-11-12 | PXD030402 | Pride
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019414 | Pride
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019415 | Pride
Project description:The PAQosome is composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and the RUVBL1/RUVBL2 AAA+ ATPases. Interestingly, NOPCHAP1 binds several RUVBL1/2 mutants except those unable to hydrolyze ATP. Moreover, while it robustly binds both yeast and human NOP58, it makes only weak interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Transient expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that select NOP58 to promote box C/D snoRNP assembly.
2020-12-14 | PXD019390 | Pride