SILAC APMS analysis of SRP72 interactome in U2OS cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Franck Vandermoere
LAB HEAD: Severine Massenet
PROVIDER: PXD042191 | Pride | 2024-06-07
REPOSITORIES: Pride
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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: SRP14, 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-SRP14 and performed stable isotope labeling by amino acids in cell culture (SILAC) proteomic experiment using GFP-SRP14 as bait.
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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.
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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.
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