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1.
Cell ; 172(5): 910-923.e16, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29474919

RESUMO

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 5' extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced.


Assuntos
Meiose/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Modelos Biológicos , Anotação de Sequência Molecular , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo
2.
Cell ; 174(3): 744-757.e24, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-29887377

RESUMO

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.


Assuntos
Núcleo Celular/ultraestrutura , Mapeamento Cromossômico/métodos , Cromossomos/fisiologia , Nucléolo Celular , Núcleo Celular/fisiologia , Cromossomos/genética , DNA/fisiologia , Eucariotos , Genoma/genética , Genoma/fisiologia , Humanos , Relação Estrutura-Atividade
3.
Cell ; 173(1): 90-103.e19, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29551269

RESUMO

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.


Assuntos
Anemia de Diamond-Blackfan/patologia , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Anemia de Diamond-Blackfan/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Mutação de Sentido Incorreto , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Cell ; 162(3): 675-86, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26189680

RESUMO

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.


Assuntos
Sistemas CRISPR-Cas , Técnicas Genéticas , Imunidade Inata , Animais , Células da Medula Óssea/imunologia , Diferenciação Celular , Sobrevivência Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
5.
Cell ; 159(7): 1698-710, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25497548

RESUMO

Cells control dynamic transitions in transcript levels by regulating transcription, processing, and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level; editing sites; and transcription, processing, and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Noncanonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one noncanonical strategy. Applying DRiLL to the regulation of noncoding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and posttranscriptional events that control dynamic changes in transcript levels using RNA sequencing data.


Assuntos
Simulação por Computador , Células Dendríticas/metabolismo , Análise de Sequência de RNA/métodos , Animais , Perfilação da Expressão Gênica/métodos , Cinética , Lipopolissacarídeos/metabolismo , Camundongos , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA não Traduzido/metabolismo , Transcrição Gênica , Tristetraprolina/metabolismo , Peixe-Zebra/embriologia
6.
Cell ; 159(1): 148-162, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25219674

RESUMO

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.


Assuntos
Pseudouridina/análise , RNA Mensageiro/química , RNA não Traduzido/química , Animais , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Perfilação da Expressão Gênica , Humanos , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pseudouridina/metabolismo , RNA/química , RNA/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Telomerase/química , Telomerase/genética
7.
Cell ; 159(2): 440-55, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25263330

RESUMO

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.


Assuntos
Adenocarcinoma/genética , Modelos Animais de Doenças , Genes Supressores de Tumor , Engenharia Genética/métodos , Neoplasias Pulmonares/genética , Oncogenes , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Dendríticas/metabolismo , Técnicas de Introdução de Genes , Vetores Genéticos , Lentivirus , Camundongos , Camundongos Transgênicos
8.
Cell ; 155(6): 1409-21, 2013 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-24269006

RESUMO

N(6)-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification.


Assuntos
Meiose , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces/citologia , Saccharomyces/metabolismo , Adenosina/análogos & derivados , Adenosina/análise , Adenosina/metabolismo , Nucléolo Celular/metabolismo , Genoma Fúngico , Metilação , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , tRNA Metiltransferases/metabolismo
9.
Genes Dev ; 34(3-4): 209-225, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31919192

RESUMO

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.


Assuntos
Aurora Quinase B/metabolismo , Cinetocoros/metabolismo , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microtúbulos/metabolismo , Proteólise
10.
EMBO J ; 42(23): e113332, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37921330

RESUMO

Amyloid-like protein assemblies have been associated with toxic phenotypes because of their repetitive and stable structure. However, evidence that cells exploit these structures to control function and activity of some proteins in response to stimuli has questioned this paradigm. How amyloid-like assembly can confer emergent functions and how cells couple assembly with environmental conditions remains unclear. Here, we study Rim4, an RNA-binding protein that forms translation-repressing assemblies during yeast meiosis. We demonstrate that in its assembled and repressive state, Rim4 binds RNA more efficiently than in its monomeric and idle state, revealing a causal connection between assembly and function. The Rim4-binding site location within the transcript dictates whether the assemblies can repress translation, underscoring the importance of the architecture of this RNA-protein structure for function. Rim4 assembly depends exclusively on its intrinsically disordered region and is prevented by the Ras/protein kinase A signaling pathway, which promotes growth and suppresses meiotic entry in yeast. Our results suggest a mechanism whereby cells couple a functional protein assembly with a stimulus to enforce a cell fate decision.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Meiose , Proteínas Amiloidogênicas/metabolismo , RNA/metabolismo , Nutrientes , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
11.
EMBO J ; 42(11): e112721, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37070548

RESUMO

Different mutations in the RNA-binding protein Pumilio1 (PUM1) cause divergent phenotypes whose severity tracks with dosage: a mutation that reduces PUM1 levels by 25% causes late-onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Yet PUM1 targets are derepressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1's RNA-binding ability. We therefore considered the possibility that the severe mutation might disrupt PUM1 interactions, and identified PUM1 interactors in the murine brain. We find that mild PUM1 loss derepresses PUM1-specific targets, but the severe mutation disrupts interactions with several RNA-binding proteins and the regulation of their targets. In patient-derived cell lines, restoring PUM1 levels restores these interactors and their targets to normal levels. Our results demonstrate that dosage sensitivity does not always signify a linear relationship with protein abundance but can involve distinct mechanisms. We propose that to understand the functions of RNA-binding proteins in a physiological context will require studying their interactions as well as their targets.


Assuntos
Encéfalo , Proteínas de Ligação a RNA , Animais , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Mutação , Encéfalo/metabolismo , Convulsões
12.
Mol Cell ; 73(1): 36-47.e10, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30503772

RESUMO

Levels of the ribosome, the conserved molecular machine that mediates translation, are tightly linked to cellular growth rate. In humans, ribosomopathies are diseases associated with cell-type-specific pathologies and reduced ribosomal protein (RP) levels. Because gene expression defects resulting from ribosome deficiency have not yet been experimentally defined, we systematically probed mRNA, translation, and protein signatures that were either unlinked from or linked to cellular growth rate in RP-deficient yeast cells. Ribosome deficiency was associated with altered translation of gene subclasses, and profound general secondary effects of RP loss on the spectrum of cellular mRNAs were seen. Among these effects, growth-defective 60S mutants increased synthesis of proteins involved in proteasome-mediated degradation, whereas 40S mutants accumulated mature 60S subunits and increased translation of ribosome biogenesis genes. These distinct signatures of protein synthesis suggest intriguing and currently mysterious differences in the cellular consequences of deficiency for small and large ribosomal subunits.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcriptoma , Proliferação de Células , Mutação , Processamento de Proteína Pós-Traducional , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo
13.
Proc Natl Acad Sci U S A ; 120(9): e2221109120, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36812203

RESUMO

Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.


Assuntos
RNA Longo não Codificante , Animais , Humanos , RNA Longo não Codificante/metabolismo , Transcrição Gênica , DNA Ribossômico/genética , Nucléolo Celular/metabolismo , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , Proteínas Ribossômicas/metabolismo , RNA não Traduzido/metabolismo , RNA Ribossômico/genética , Proteínas de Transporte/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo
14.
Nature ; 556(7702): 501-504, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29670287

RESUMO

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Proteínas I-kappa B/metabolismo , Succinatos/metabolismo , Animais , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Psoríase/tratamento farmacológico , Psoríase/patologia , Estresse Fisiológico/efeitos dos fármacos , Succinatos/administração & dosagem , Succinatos/química , Succinatos/farmacologia , Succinatos/uso terapêutico , Receptores Toll-Like/imunologia
15.
Trends Biochem Sci ; 44(2): 95-109, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30473427

RESUMO

The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years. Recent advances in mass spectrometry (MS) and high-throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast and mammalian systems. In addition to RP stoichiometry, ribosomes host a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible that these proteoforms combine to function as a 'ribosome code' to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the ribosome code.


Assuntos
Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Animais , Ensaios de Triagem em Larga Escala , Humanos , Espectrometria de Massas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo
16.
Trends Biochem Sci ; 44(5): 478-479, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30792028

RESUMO

Contrary to the textbook model, recent measurements demonstrated unexpected diversity in ribosomal composition that likely enables specialized translational functions. Methods based on liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) enable direct quantification of ribosomal proteins with high specificity, accuracy, and throughput. LC-MS/MS can be 'top-down', analyzing intact proteins, or more commonly 'bottom-up', where proteins are digested to peptides prior to analysis. Changes to rRNA can be examined using either LC-MS/MS or sequencing-based approaches. The regulation of protein synthesis by specialized ribosomes can be examined by multiple methods. These include the popular 'Ribo-Seq' method for analyzing ribosome density on a given mRNA, as well as LC-MS/MS approaches incorporating pulse-labelling with stable isotopes (SILAC) to monitor protein synthesis and degradation.


Assuntos
RNA Ribossômico/química , Ribossomos/química , Cromatografia Líquida , Modelos Moleculares , Proteínas/química , Espectrometria de Massas em Tandem
17.
Chembiochem ; 24(10): e202200706, 2023 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-36893077

RESUMO

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.


Assuntos
Proteínas Tirosina Fosfatases , Tirosina , Domínio Catalítico , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo
18.
Mol Cell ; 60(5): 816-827, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26638175

RESUMO

A fundamental goal of genomics is to identify the complete set of expressed proteins. Automated annotation strategies rely on assumptions about protein-coding sequences (CDSs), e.g., they are conserved, do not overlap, and exceed a minimum length. However, an increasing number of newly discovered proteins violate these rules. Here we present an experimental and analytical framework, based on ribosome profiling and linear regression, for systematic identification and quantification of translation. Application of this approach to lipopolysaccharide-stimulated mouse dendritic cells and HCMV-infected human fibroblasts identifies thousands of novel CDSs, including micropeptides and variants of known proteins, that bear the hallmarks of canonical translation and exhibit translation levels and dynamics comparable to that of annotated CDSs. Remarkably, many translation events are identified in both mouse and human cells even when the peptide sequence is not conserved. Our work thus reveals an unexpected complexity to mammalian translation suited to provide both conserved regulatory or protein-based functions.


Assuntos
Proteoma/metabolismo , Proteômica/métodos , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Sequência Conservada , Células Dendríticas/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Fases de Leitura Aberta , Análise de Regressão
19.
Mol Cell Proteomics ; 20: 100016, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33556866

RESUMO

In all cells, proteins are continuously synthesized and degraded to maintain protein homeostasis and modify gene expression levels in response to stimuli. Collectively, the processes of protein synthesis and degradation are referred to as protein turnover. At a steady state, protein turnover is constant to maintain protein homeostasis, but in dynamic responses, proteins change their rates of synthesis and degradation to adjust their proteomes to internal or external stimuli. Thus, probing the kinetics and dynamics of protein turnover lends insight into how cells regulate essential processes such as growth, differentiation, and stress response. Here, we outline historical and current approaches to measuring the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable isotope-labeled amino acids. We highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigation.


Assuntos
Proteoma , Aminoácidos , Animais , Humanos , Marcação por Isótopo , Luz , Preparações Farmacêuticas , Proteômica , Radioisótopos
20.
Medicina (Kaunas) ; 58(12)2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36556899

RESUMO

Background and Objectives: The vaccine against human papilloma virus (HPV) infection is recommended, according to the Serbian National Immunization Program, for children and adolescents aged 9−19 years. Three doses are given keeping in mind the recommendation that the second dose should be administered at least one month after the first dose, and the third at least three months after the second dose. No children who participated in this first study received the third dose because they did not meet these criteria. The study explored parents' knowledge about HPV infection and their awareness of the HPV vaccine. Materials and Methods: A cross-sectional questionnaire-based study was carried out in the city of Nis, in southeastern Serbia. According to the 2011 population census, the sample of children aged 9 to 19 was 850, and during the observed period, 631 children received the vaccine. A total of 615 fully completed questionnaires filled out by parents were included in the study. The study was carried out from 6 June 2022 to 7 October 2022. Multivariable logistic regression analysis was used. The odds ratio (OR) and 95% confidence intervals (CI) were calculated. The statistical significance was p < 0.05. Results: A total of 615 children were included in the study (499 were vaccinated with the first dose and 116 with the second). Out of 499 children vaccinated with the first dose, 398 (79.6%) were girls, which is significantly higher than the rate for boys (101). The independent variable sex was statistically significant at the level of p = 0.84, OR = 2.664 (95% CI from 0.879 to 7.954). Boys are 164% less likely to be vaccinated with the HPV vaccine than girls. We determined that the independent variable place of residence was significant at the level of p = 0.041, (OR = 3.809, 95% CI from 1.702 to 8.525). Based on these findings, we determined that parents who came from rural areas were 82% less likely to know about HPV infection and HPV vaccination. Children under 15 years of age were significantly more vaccinated than those ≥15 years (OR = 3.698, 95% CI from 1.354 to 12.598). The independent variable parental education was significant at the level of OR = 0.494, 95% CI from 0.301 to 0.791. Parents who had medical education showed significantly higher awareness about the infection caused by HPV and about the HPV vaccine (p = 0.004) than parents with no medical education. The possibility that a parent would decide to vaccinate a child significantly increased upon a pediatrician's recommendation, p = 0.000 with OR = 0.250 (95% CI from 0.127 to 0.707). Health insurance coverage of HPV vaccination for children aged 9−19 years significantly increased the probability of a positive parental decision to vaccinate a child, p = 0.001 with OR = 3.034 (95% CI from 1.063 to 8.662). Conclusion: We identified several significant factors that were important for HPV vaccination such as: children under 15 years, female sex, urban place of residence, medical education of parents, pediatrician's recommendation of the HPV vaccination, and HPV vaccination free of charge. Health education and the promotion of HPV vaccination as well as healthy sexual behavior are important factors in the preservation and improvement of the health of the whole population.


Assuntos
Infecções por Papillomavirus , Vacinas contra Papillomavirus , Masculino , Adolescente , Humanos , Feminino , Infecções por Papillomavirus/prevenção & controle , Sérvia , Vacinas contra Papillomavirus/uso terapêutico , Estudos Transversais , Conhecimentos, Atitudes e Prática em Saúde , Pais , Inquéritos e Questionários
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