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1.
MicroPubl Biol ; 20222022.
Artigo em Inglês | MEDLINE | ID: mdl-36212518

RESUMO

RNA helicases are involved in nearly all aspects of RNA metabolism and factor prominently in ribosome assembly. The SSU processome includes 10 helicases and many helicase-cofactors. Together, they mediate the structural rearrangements that occur as part of ribosomal SSU assembly. During the identification of the SSU processome component Utp25/Def, it was noticed that the protein displays some sequence similarity to DEAD-box RNA helicases and is essential for growth. Interestingly, mutational ablation showed that Utp25's DEAD-box motifs are dispensable. Here, we show that the Utp25 AlphaFold prediction displays considerable structural similarity to DEAD-box helicases and is the first fully validated pseudohelicase.

2.
Am J Physiol Regul Integr Comp Physiol ; 323(3): R319-R330, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35670765

RESUMO

The peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) is central to the regulation of cellular and mitochondrial energy homeostasis in mammals, but its role in other vertebrates remains unclear. Indeed, previous work suggests extensive structural and functional divergence of PGC-1α in teleosts but this remains to be directly tested. Here, we describe the initial characterization of heterozygous PGC-1α mutant zebrafish lines created by CRISPR-Cas9 disruptions of an evolutionarily conserved regulatory region of the PGC-1α proximal promoter. Using qPCR, we confirmed the disruption of PGC-1α gene expression in striated muscle, leading to a simultaneous fourfold increase in mixed skeletal muscle PGC-1α mRNA levels and an opposite fourfold downregulation in cardiac muscle. In mixed skeletal muscle, most downstream effector genes were largely unaffected yet two mitochondrial lipid transporters, carnitine palmitoyltransferase-1 and -2, were strongly induced. Conversely, PGC-1α depression in cardiac muscle reduced the expression of several transcriptional regulators (estrogen-related receptor α, nuclear respiratory factor 1, and PGC-1ß) without altering metabolic gene expression. Using high-resolution respirometry, we determined that white muscle exhibited increased lipid oxidative capacity with little difference in markers of mitochondrial abundance. Finally, using whole animal intermittent respirometry, we show that mutant fish exhibit a twofold higher basal metabolism than their wild-type counterparts. Altogether, this new model confirms a central but complex role for PGC-1α in mediating energy utilization in zebrafish, and we propose its use as a valuable tool to explore the intricate regulatory pathways of energy homeostasis in a popular biomedical model.


Assuntos
Músculo Esquelético , Peixe-Zebra , Animais , Metabolismo Energético/genética , Lipídeos , Mamíferos/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
3.
RNA ; 24(1): 77-89, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29054886

RESUMO

Ribosome assembly is an evolutionarily conserved and energy intensive process required for cellular growth, proliferation, and maintenance. In yeast, assembly of the small ribosomal subunit (SSU) requires approximately 75 assembly factors that act in coordination to form the SSU processome, a 6 MDa ribonucleoprotein complex. The SSU processome is required for processing, modifying, and folding the preribosomal RNA (rRNA) to prepare it for incorporation into the mature SSU. Although the protein composition of the SSU processome has been known for some time, the interaction network of the proteins required for its assembly has remained poorly defined. Here, we have used a semi-high-throughput yeast two-hybrid (Y2H) assay and coimmunoprecipitation validation method to produce a high-confidence interactome of SSU processome assembly factors (SPAFs), providing essential insight into SSU assembly and ribosome biogenesis. Further, we used glycerol density-gradient sedimentation to reveal the presence of protein subcomplexes that have not previously been observed. Our work not only provides essential insight into SSU assembly and ribosome biogenesis, but also serves as an important resource for future investigations into how defects in biogenesis and assembly cause congenital disorders of ribosomes known as ribosomopathies.


Assuntos
Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos Multiproteicos/metabolismo , Mapas de Interação de Proteínas , Ribossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido
4.
Genes Dev ; 29(8): 862-75, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25877921

RESUMO

Maturation of the large ribosomal subunit (LSU) in eukaryotes is a complex and highly coordinated process that requires the concerted action of a large, dynamic, ribonucleoprotein complex, the LSU processome. While we know that >80 ribosome biogenesis factors are required throughout the course of LSU assembly, little is known about how these factors interact with each other within the LSU processome. To interrogate its organization and architecture, we took a systems biology approach and performed a semi-high-throughput, array-based, directed yeast two-hybrid assay. Assaying 4800 protein-protein interactions, we identified 232 high-confidence, binary-interacting protein pairs, representing a fourfold increase from current knowledge. The resulting LSU processome interactome map has enhanced our understanding of the organization and function of the biogenesis factors within the LSU processome, revealing both novel and previously identified subcomplexes and hub proteins, including Nop4.


Assuntos
Mapas de Interação de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido
5.
Cell Rep ; 2(2): 372-85, 2012 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-22902402

RESUMO

Eukaryotic ribosome biogenesis requires hundreds of trans-acting factors and dozens of RNAs. Although most factors required for ribosome biogenesis have been identified, little is known about their regulation. Here, we reveal that the yeast deubiquitinating enzyme Ubp10 is localized to the nucleolus and that ubp10Δ cells have reduced pre-rRNAs, mature rRNAs, and translating ribosomes. Through proteomic analyses, we found that Ubp10 interacts with proteins that function in rRNA production and ribosome biogenesis. In particular, we discovered that the largest subunit of RNA polymerase I (RNAPI) is stabilized via Ubp10-mediated deubiquitination and that this is required in order to achieve optimal levels of ribosomes and cell growth. USP36, the human ortholog of Ubp10, complements the ubp10Δ allele for RNAPI stability, pre-rRNA processing, and cell growth in yeast, suggesting that deubiquitination of RNAPI may be conserved in eukaryotes. Our work implicates Ubp10/USP36 as a key regulator of rRNA production through control of RNAPI stability.


Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase I/metabolismo , RNA Fúngico/biossíntese , RNA Ribossômico/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterase/metabolismo , Nucléolo Celular/genética , Estabilidade Enzimática/fisiologia , Teste de Complementação Genética , Humanos , Proteínas Nucleares/genética , RNA Polimerase I/genética , RNA Fúngico/genética , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina Tiolesterase/genética , Ubiquitinação/fisiologia
6.
PLoS One ; 6(3): e17701, 2011 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-21423703

RESUMO

BACKGROUND: The small subunit (SSU) processome is a large ribonucleoprotein complex involved in small ribosomal subunit assembly. It consists of the U3 snoRNA and ∼72 proteins. While most of its components have been identified, the protein-protein interactions (PPIs) among them remain largely unknown, and thus the assembly, architecture and function of the SSU processome remains unclear. METHODOLOGY: We queried PPI databases for SSU processome proteins to quantify the degree to which the three genome-wide high-throughput yeast two-hybrid (HT-Y2H) studies, the genome-wide protein fragment complementation assay (PCA) and the literature-curated (LC) datasets cover the SSU processome interactome. CONCLUSIONS: We find that coverage of the SSU processome PPI network is remarkably sparse. Two of the three HT-Y2H studies each account for four and six PPIs between only six of the 72 proteins, while the third study accounts for as little as one PPI and two proteins. The PCA dataset has the highest coverage among the genome-wide studies with 27 PPIs between 25 proteins. The LC dataset was the most extensive, accounting for 34 proteins and 38 PPIs, many of which were validated by independent methods, thereby further increasing their reliability. When the collected data were merged, we found that at least 70% of the predicted PPIs have yet to be determined and 26 proteins (36%) have no known partners. Since the SSU processome is conserved in all Eukaryotes, we also queried HT-Y2H datasets from six additional model organisms, but only four orthologues and three previously known interologous interactions were found. This provides a starting point for further work on SSU processome assembly, and spotlights the need for a more complete genome-wide Y2H analysis.


Assuntos
Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Subunidades Ribossômicas Menores/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Mineração de Dados , Genoma Fúngico/genética , Humanos , Análise de Componente Principal , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido
7.
Wiley Interdiscip Rev RNA ; 2(1): 1-21, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21318072

RESUMO

The small subunit (SSU) processome is a 2.2-MDa ribonucleoprotein complex involved in the processing, assembly, and maturation of the SSU of eukaryotic ribosomes. The identities of many of the factors involved in SSU biogenesis have been elucidated over the past 40 years. However, as our understanding increases, so do the number of questions about the nature of this complicated process. Cataloging the components is the first step toward understanding the molecular workings of a system. This review will focus on how identifying components of ribosome biogenesis has led to the knowledge of how these factors, protein and RNA alike, associate with one another into subcomplexes, with a concentration on the small ribosomal subunit. We will also explore how this knowledge of subcomplex assembly has informed our understanding of the workings of the ribosome synthesis system as a whole.


Assuntos
Modificação Traducional de Proteínas , Ribonucleoproteínas/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Ribossomos/metabolismo , Animais , Eucariotos/genética , Eucariotos/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Modificação Traducional de Proteínas/genética , RNA Ribossômico 18S/química , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas/química
8.
RNA ; 16(11): 2156-69, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20884785

RESUMO

The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A0, A1, and A2. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature-sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.


Assuntos
Proteínas de Transporte/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Deleção de Genes , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Especificidade por Substrato
9.
BMC Genomics ; 10: 528, 2009 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-19917113

RESUMO

BACKGROUND: U3 snoRNA is a box C/D small nucleolar RNA (snoRNA) involved in the processing events that liberate 18S rRNA from the ribosomal RNA precursor (pre-rRNA). Although U3 snoRNA is present in all eukaryotic organisms, most investigations of it have focused on fungi (particularly yeasts), animals and plants. Relatively little is known about U3 snoRNA and its gene(s) in the phylogenetically broad assemblage of protists (mostly unicellular eukaryotes). In the euglenozoon Euglena gracilis, a distant relative of the kinetoplastid protozoa, Southern analysis had previously revealed at least 13 bands hybridizing with U3 snoRNA, suggesting the existence of multiple copies of U3 snoRNA genes. RESULTS: Through screening of a lambda genomic library and PCR amplification, we recovered 14 U3 snoRNA gene variants, defined by sequence heterogeneities that are mostly located in the U3 3'-stem-loop domain. We identified three different genomic arrangements of Euglena U3 snoRNA genes: i) stand-alone, ii) linked to tRNAArg genes, and iii) linked to a U5 snRNA gene. In arrangement ii), the U3 snoRNA gene is positioned upstream of two identical tRNAArg genes that are convergently transcribed relative to the U3 gene. This scenario is reminiscent of a U3 snoRNA-tRNA gene linkage previously described in trypanosomatids. We document here twelve different U3 snoRNA-U5 snRNA gene arrangements in Euglena; in each case, the U3 gene is linked to a downstream and convergently oriented U5 gene, with the intergenic region differing in length and sequence among the variants. CONCLUSION: The multiple U3 snoRNA-U5 snRNA gene linkages, which cluster into distinct families based on sequence similarities within the intergenic spacer, presumably arose by genome, chromosome, and/or locus duplications. We discuss possible reasons for the existence of the unusually large number of U3 snoRNA genes in the Euglena genome. Variability in the signal intensities of the multiple Southern hybridization bands raises the possibility that Euglena contains a naturally aneuploid chromosome complement.


Assuntos
Euglena gracilis/genética , Dosagem de Genes , Ligação Genética , RNA Nuclear Pequeno/genética , RNA Nucleolar Pequeno/genética , Sequência de Bases , DNA de Protozoário/genética , Evolução Molecular , Genoma de Protozoário/genética , Genômica , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , RNA de Transferência/genética , Trypanosomatina/genética
10.
Nature ; 443(7113): 863-6, 2006 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17051219

RESUMO

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs. It was first identified and characterized in animals, where it was found to contain several unique RNA constituents that share structural similarity with and seem to be functionally analogous to the small nuclear RNAs (snRNAs) contained in the major spliceosome. Subsequently, minor spliceosomal components and U12-type introns have been found in plants but not in fungi. Unlike that of the major spliceosome, which arose early in the eukaryotic lineage, the evolutionary history of the minor spliceosome is unclear because there is evidence of it in so few organisms. Here we report the identification of homologues of minor-spliceosome-specific proteins and snRNAs, and U12-type introns, in distantly related eukaryotic microbes (protists) and in a fungus (Rhizopus oryzae). Cumulatively, our results indicate that the minor spliceosome had an early origin: several of its characteristic constituents are present in representative organisms from all eukaryotic supergroups for which there is any substantial genome sequence information. In addition, our results reveal marked evolutionary conservation of functionally important sequence elements contained within U12-type introns and snRNAs.


Assuntos
Acanthamoeba castellanii/genética , Evolução Molecular , Rhizopus/genética , Spliceossomos/química , Spliceossomos/genética , Acanthamoeba castellanii/química , Animais , Sequência de Bases , Células Eucarióticas/metabolismo , Etiquetas de Sequências Expressas , Humanos , Íntrons/genética , Dados de Sequência Molecular , Filogenia , Splicing de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Rhizopus/química , Spliceossomos/metabolismo
11.
Nucleic Acids Res ; 33(9): 2781-91, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15894796

RESUMO

Box C/D ribonucleoprotein (RNP) particles mediate O2'-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.


Assuntos
Proteínas Cromossômicas não Histona/genética , Euglena gracilis/genética , Evolução Molecular , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas/genética , Animais , Sequência de Bases , Proteínas Cromossômicas não Histona/química , DNA Complementar/química , Células Eucarióticas/química , Componentes do Gene , Íntrons , Dados de Sequência Molecular , RNA Nuclear Pequeno/química , Ribonucleoproteínas/química , Alinhamento de Sequência
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