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1.
Nucleic Acids Res ; 51(7): 3357-3374, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-36869663

RESUMO

The conserved H/ACA RNPs consist of one H/ACA RNA and 4 core proteins: dyskerin, NHP2, NOP10, and GAR1. Its assembly requires several assembly factors. A pre-particle containing the nascent RNAs, dyskerin, NOP10, NHP2 and NAF1 is assembled co-transcriptionally. NAF1 is later replaced by GAR1 to form mature RNPs. In this study, we explore the mechanism leading to the assembly of H/ACA RNPs. We performed the analysis of GAR1, NHP2, SHQ1 and NAF1 proteomes by quantitative SILAC proteomic, and analyzed purified complexes containing these proteins by sedimentation on glycerol gradient. We propose the formation of several distinct intermediate complexes during H/ACA RNP assembly, notably the formation of early protein-only complexes containing at least the core proteins dyskerin, NOP10, and NHP2, and the assembly factors SHQ1 and NAF1. We also identified new proteins associated with GAR1, NHP2, SHQ1 and NAF1, which can be important for box H/ACA assembly or function. Moreover, even though GAR1 is regulated by methylations, the nature, localization, and functions of these methylations are not well known. Our MS analysis of purified GAR1 revealed new sites of arginine methylations. Additionally, we showed that unmethylated GAR1 is correctly incorporated in H/ACA RNPs, even though with less efficiency than methylated ones.


Assuntos
Proteômica , Ribonucleoproteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Ligação a RNA , RNA/genética
2.
Nucleic Acids Res ; 43(18): 8973-89, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26275778

RESUMO

The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins.


Assuntos
Proteínas do Olho/metabolismo , Chaperonas Moleculares/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas do Complexo SMN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Linhagem Celular , Corpos Enovelados/metabolismo , Citoplasma/metabolismo , Proteínas do Olho/química , Proteínas do Olho/genética , Células HeLa , Humanos , Mutagênese Insercional , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética
3.
Nucleic Acids Res ; 41(2): 1255-72, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23221635

RESUMO

Spinal muscular atrophy is a severe motor neuron disease caused by reduced levels of the ubiquitous Survival of MotoNeurons (SMN) protein. SMN is part of a complex that is essential for spliceosomal UsnRNP biogenesis. Signal recognition particle (SRP) is a ribonucleoprotein particle crucial for co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. SRP biogenesis is a nucleo-cytoplasmic multistep process in which the protein components, except SRP54, assemble with 7S RNA in the nucleolus. Then, SRP54 is incorporated after export of the pre-particle into the cytoplasm. The assembly factors necessary for SRP biogenesis remain to be identified. Here, we show that 7S RNA binds to purified SMN complexes in vitro and that SMN complexes associate with SRP in cellular extracts. We identified the RNA determinants required. Moreover, we report a specific reduction of 7S RNA levels in the spinal cord of SMN-deficient mice, and in a Schizosaccharomyces pombe strain carrying a temperature-degron allele of SMN. Additionally, microinjected antibodies directed against SMN or Gemin2 interfere with the association of SRP54 with 7S RNA in Xenopus laevis oocytes. Our data show that reduced levels of the SMN protein lead to defect in SRP steady-state level and describe the SMN complex as the first identified cellular factor required for SRP biogenesis.


Assuntos
RNA Citoplasmático Pequeno/metabolismo , Proteínas do Complexo SMN/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Alelos , Animais , Anticorpos/farmacologia , Sequência de Bases , Citoplasma/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Atrofia Muscular Espinal/metabolismo , Mutação , RNA Citoplasmático Pequeno/química , RNA Nuclear Pequeno/metabolismo , Proteínas do Complexo SMN/antagonistas & inibidores , Proteínas do Complexo SMN/imunologia , Schizosaccharomyces/genética , Partícula de Reconhecimento de Sinal/química , Medula Espinal/metabolismo , Xenopus laevis
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