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1.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38858088

RESUMO

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.


Assuntos
Nucléolo Celular , Proteômica , Ribossomos , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/metabolismo , Nucléolo Celular/metabolismo , Ribossomos/metabolismo , Humanos , Proteômica/métodos , Proteínas Nucleares/metabolismo , Corpos Enovelados/metabolismo , Células HeLa , Retículo Endoplasmático/metabolismo
2.
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
3.
Nat Commun ; 12(1): 1859, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767140

RESUMO

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ativação Transcricional/fisiologia , Proliferação de Células/fisiologia , Cromatina/genética , Cristalografia por Raios X , Histonas/metabolismo , Ressonância Magnética Nuclear Biomolecular , RNA Polimerase II/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica/genética
5.
Biochimie ; 164: 99-104, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30978374

RESUMO

The signal recognition particle (SRP) is a universally conserved non-coding ribonucleoprotein complex that is essential for targeting transmembrane and secretory proteins to the endoplasmic reticulum. Its composition and size varied during evolution. In mammals, SRP contains one RNA molecule, 7SL RNA, and six proteins: SRP9, 14, 19, 54, 68 and 72. Despite a very good understanding of the SRP structure and of the SRP assembly in vitro, how SRP is assembled in vivo remains largely enigmatic. Here we review current knowledge on how the 7SL RNA is assembled with core proteins to form functional RNP particles in cells. SRP biogenesis is believed to take place both in the nucleolus and in the cytoplasm and to rely on the survival of motor neuron complex, whose defect leads to spinal muscular atrophy.


Assuntos
Células Eucarióticas/metabolismo , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Animais , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas do Complexo SMN/metabolismo
6.
RNA ; 25(4): 496-506, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30700579

RESUMO

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins (C/D snoRNPs) is guided by conserved trans-acting factors that act collectively to assemble the core proteins SNU13/Snu13, NOP58/Nop58, NOP56/Nop56, FBL/Nop1, and box C/D small nucleolar RNAs (C/D snoRNAs), in human and in yeast, respectively. This finely elaborated process involves the sequential interplay of snoRNP-related proteins and RNA through the formation of transient pre-RNP complexes. BCD1/Bcd1 protein is essential for yeast cell growth and for the specific accumulation of box C/D snoRNAs. In this work, chromatin, RNA, and protein immunoprecipitation assays revealed the ordered loading of several snoRNP-related proteins on immature and mature snoRNA species. Our results identify Bcd1p as an assembly factor of C/D snoRNP biogenesis that is likely recruited cotranscriptionally and that directs the loading of the core protein Nop58 on RNA.


Assuntos
Fator 6 Semelhante a Kruppel/genética , Proteínas Nucleares/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Humanos , Fator 6 Semelhante a Kruppel/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica
7.
Nucleic Acids Res ; 45(9): 5399-5413, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28115638

RESUMO

Selenoprotein synthesis requires the co-translational recoding of a UGASec codon. This process involves an RNA structural element, called Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2). Several selenoprotein mRNAs undergo unusual cap hypermethylation by the trimethylguanosine synthase 1 (Tgs1), which is recruited by the ubiquitous Survival of MotoNeurons (SMN) protein. SMN, the protein involved in spinal muscular atrophy, is part of a chaperone complex that collaborates with the methylosome for RNP assembly. Here, we analyze the role of individual SMN and methylosome components in selenoprotein mRNP assembly and translation. We show that SBP2 interacts directly with four proteins of the SMN complex and the methylosome core proteins. Nevertheless, SBP2 is not a methylation substrate of the methylosome. We found that both SMN and methylosome complexes are required for efficient translation of the selenoprotein GPx1 in vivo. We establish that the steady-state level of several selenoprotein mRNAs, major regulators of oxidative stress damage in neurons, is specifically reduced in the spinal cord of SMN-deficient mice and that cap hypermethylation of GPx1 mRNA is affected. Altogether we identified a new function of the SMN complex and the methylosome in selenoprotein mRNP assembly and expression.


Assuntos
Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas do Complexo SMN/metabolismo , Selenoproteínas/metabolismo , Glutationa Peroxidase , Células HEK293 , Células HeLa , Humanos , Metilação , Modelos Biológicos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Ligação Proteica , Medula Espinal/metabolismo , Glutationa Peroxidase GPX1
8.
RNA Biol ; 14(6): 680-692, 2017 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27715451

RESUMO

Box C/D and box H/ACA snoRNAs are abundant non-coding RNAs that localize in the nucleolus and mostly function as guides for nucleotide modifications. While a large pool of snoRNAs modifies rRNAs, an increasing number of snoRNAs could also potentially target mRNAs. ScaRNAs belong to a family of specific RNAs that localize in Cajal bodies and that are structurally similar to snoRNAs. Most scaRNAs are involved in snRNA modification, while telomerase RNA, which contains H/ACA motifs, functions in telomeric DNA synthesis. In this review, we describe how box C/D and H/ACA snoRNAs are processed and assembled with core proteins to form functional RNP particles. Their biogenesis involve several transport factors that first direct pre-snoRNPs to Cajal bodies, where some processing steps are believed to take place, and then to nucleoli. Assembly of core proteins involves the HSP90/R2TP chaperone-cochaperone system for both box C/D and H/ACA RNAs, but also several factors specific for each family. These assembly factors chaperone unassembled core proteins, regulate the formation and disassembly of pre-snoRNP intermediates, and control the activity of immature particles. The AAA+ ATPase RUVBL1 and RUVBL2 belong to the R2TP co-chaperones and play essential roles in snoRNP biogenesis, as well as in the formation of other macro-molecular complexes. Despite intensive research, their mechanisms of action are still incompletely understood.


Assuntos
RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Animais , Proteínas de Transporte , Corpos Enovelados/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico , Processamento Pós-Transcricional do RNA , RNA Nucleolar Pequeno/química , Transdução de Sinais , Transcrição Gênica
9.
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
10.
Nucleic Acids Res ; 42(16): 10731-47, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25170085

RESUMO

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82-R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3'-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317-352-Hit1p70-164 complex reveals a novel mode of protein-protein association explaining the strong stability of the Rsa1p-Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p-Rsa1p-Hit1p heterotrimer.


Assuntos
RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento de Terminações 3' de RNA , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
11.
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
12.
J Biol Chem ; 283(9): 5598-610, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18093976

RESUMO

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Hipotálamo/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Éxons/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Humanos , Hipotálamo/patologia , Camundongos , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor
13.
RNA ; 12(8): 1583-93, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16804160

RESUMO

Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Psi formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Psi26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5' and/or 3' extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.


Assuntos
Hidroliases/metabolismo , RNA Fúngico/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Animais , Hidroliases/genética , Técnicas In Vitro , Camundongos , Conformação de Ácido Nucleico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA de Transferência/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
14.
J Biol Chem ; 279(51): 52998-3006, 2004 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-15466869

RESUMO

Cytoplasmic and mitochondrial tRNAs contain several pseudouridylation sites, and the tRNA:Psi-synthase acting at position 32 had not been identified in Saccharomyces cerevisiae. By combining genetic and biochemical analyses, we demonstrate that two enzymes, Rib2/Pus8p and Pus9p, are required for Psi32 formation in cytoplasmic and mitochondrial tRNAs, respectively. Pus9p acts mostly in mitochondria, and Rib2/Pus8p is strictly cytoplasmic. This is the first case reported so far of two distinct tRNA modification enzymes acting at the same position but present in two different compartments. This peculiarity may be the consequence of a gene fusion that occurred during yeast evolution. Indeed, Rib2/Pus8p displays two distinct catalytic activities involved in completely unrelated metabolism: its C-terminal domain has a DRAP-deaminase activity required for riboflavin biogenesis in the cytoplasm, whereas its N-terminal domain carries the tRNA:Psi32-synthase activity. Pus9p has only a tRNA:Psi32-synthase activity and contains a characteristic mitochondrial targeting sequence at its N terminus. These results are discussed in terms of RNA:Psi-synthase evolution.


Assuntos
Aminoidrolases/fisiologia , Citoplasma/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Uridina/química , Sequência de Aminoácidos , Aminoidrolases/química , Ácido Aspártico/química , Catálise , Sistema Livre de Células , Relação Dose-Resposta a Droga , Deleção de Genes , Teste de Complementação Genética , Transferases Intramoleculares/química , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA/química , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos
15.
Mol Cell Biol ; 22(18): 6533-41, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12192051

RESUMO

The common neurodegenerative disease spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN associates with several proteins (Gemin2 to Gemin6) to form a large complex which is found both in the cytoplasm and in the nucleus. The SMN complex functions in the assembly and metabolism of several RNPs, including spliceosomal snRNPs. The snRNP core assembly takes place in the cytoplasm from Sm proteins and newly exported snRNAs. Here, we identify three distinct cytoplasmic SMN complexes, each representing a defined intermediate in the snRNP biogenesis pathway. We show that the SMN complex associates with newly exported snRNAs containing the nonphosphorylated form of the snRNA export factor PHAX. The second SMN complex identified contains assembled Sm cores and m(3)G-capped snRNAs. Finally, the SMN complex is associated with a preimport complex containing m(3)G-capped snRNP cores bound to the snRNP nuclear import mediator snurportin1. Thus, the SMN complex is associated with snRNPs during the entire process of their biogenesis in the cytoplasm and may have multiple functions throughout this process.


Assuntos
Citoplasma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , DNA/metabolismo , Células HeLa , Humanos , Substâncias Macromoleculares , Microscopia de Fluorescência , Modelos Biológicos , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas de Ligação ao Cap de RNA , Proteínas de Ligação a RNA , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas do Complexo SMN , Spliceossomos/metabolismo
16.
Curr Opin Cell Biol ; 14(3): 305-12, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12067652

RESUMO

Spinal muscular atrophy is a common, often lethal, neurodegenerative disease that results from low levels of, or loss-of-function mutations in, the SMN (survival of motor neurons) protein. SMN oligomerizes and forms a stable complex with five additional proteins: Gemins 2-6. SMN also interacts with several additional proteins referred to as "substrates". Most of these substrates contain a domain enriched in arginine and glycine residues (the RG-rich domain), and are constituents of different ribonucleoprotein complexes. Recent studies revealed that the substrates can be modified by an arginine methyltransferase complex, the methylosome. This forms symmetrical dimethylarginines within the RG-rich domains of the substrates, thereby converting them to high-affinity binders of the SMN complex, and most likely providing regulation of the ribonucleoprotein assembly processes.


Assuntos
Proteínas do Tecido Nervoso/fisiologia , Ribonucleoproteínas/biossíntese , Sequência de Aminoácidos , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Citoplasma/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Metiltransferases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/análise , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a RNA , Proteínas do Complexo SMN
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