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1.
Nucleic Acids Res ; 51(10): 5144-5161, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-37021550

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.


Assuntos
Distrofia Muscular Facioescapuloumeral , Humanos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismo , Fatores de Transcrição/metabolismo
2.
Mol Cell Proteomics ; 21(7): 100243, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35577067

RESUMO

Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumor development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl stable isotope labeling with amino acids in cell culture experiments analyzed with a machine learning-based tool and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein-RNA interactions and suggest a role in rewiring protein-protein interactions, for which we provide experimental evidence for a representative case (i.e., NONO [non-POU domain-containing octamer-binding protein]-paraspeckle component 1 [PSPC1]). Upon intersecting our R-methyl-sites dataset with the PhosphoSitePlus phosphorylation dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of heavy methyl stable isotope labeling with amino acids in cell culture to identify unconventional methylated residues and successfully identified novel histone methylation marks on serine 28 and threonine 32 of H3. The database generated, named ProMetheusDB, is freely accessible at https://bioserver.ieo.it/shiny/app/prometheusdb.


Assuntos
Processamento de Proteína Pós-Traducional , Proteoma , Aminoácidos/metabolismo , Humanos , Marcação por Isótopo/métodos , Espectrometria de Massas , Metilação , Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo
3.
Nucleic Acids Res ; 50(6): 3475-3489, 2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35244721

RESUMO

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.


Assuntos
COVID-19 , Nanoporos , RNA Guia de Cinetoplastídeos/química , COVID-19/genética , Genoma Viral/genética , Humanos , Capuzes de RNA , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética
4.
Nucleic Acids Res ; 48(1): 96-115, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31777917

RESUMO

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.


Assuntos
Epigênese Genética , MicroRNAs/genética , Proteínas do Fator Nuclear 90/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Animais , Arginina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Marcação por Isótopo , Lisina/metabolismo , Metilação , MicroRNAs/biossíntese , MicroRNAs/classificação , Proteínas do Fator Nuclear 90/metabolismo , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo
5.
Nat Chem Biol ; 11(8): 571-578, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26167872

RESUMO

The CEBPA gene is mutated in 9% of patients with acute myeloid leukemia (AML). Selective expression of a short (30-kDa) CCAAT-enhancer binding protein-α (C/EBPα) translational isoform, termed p30, represents the most common type of CEBPA mutation in AML. The molecular mechanisms underlying p30-mediated transformation remain incompletely understood. We show that C/EBPα p30, but not the normal p42 isoform, preferentially interacts with Wdr5, a key component of SET/MLL (SET-domain/mixed-lineage leukemia) histone-methyltransferase complexes. Accordingly, p30-bound genomic regions were enriched for MLL-dependent H3K4me3 marks. The p30-dependent increase in self-renewal and inhibition of myeloid differentiation required Wdr5, as downregulation of the latter inhibited proliferation and restored differentiation in p30-dependent AML models. OICR-9429 is a new small-molecule antagonist of the Wdr5-MLL interaction. This compound selectively inhibited proliferation and induced differentiation in p30-expressing human AML cells. Our data reveal the mechanism of p30-dependent transformation and establish the essential p30 cofactor Wdr5 as a therapeutic target in CEBPA-mutant AML.


Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Di-Hidropiridinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Células Tumorais Cultivadas
6.
Chemistry ; 21(28): 10116-22, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26033174

RESUMO

Phosphoanhydrides (P-anhydrides) are ubiquitously occurring modifications in nature. Nucleotides and their conjugates, for example, are among the most important building blocks and signaling molecules in cell biology. To study and manipulate their biological functions, a diverse range of analogues have been developed. Phosphate-modified analogues have been successfully applied to study proteins that depend on these abundant cellular building blocks, but very often both the preparation and purification of these molecules are challenging. This study discloses a general access to P-anhydrides, including different nucleotide probes, that greatly facilitates their preparation and isolation. The convenient and scalable synthesis of, for example, (18) O labeled nucleoside triphosphates holds promise for future applications in phosphoproteomics.


Assuntos
Anidridos/síntese química , Nucleosídeos/química , Nucleotídeos/química , Fosfatos/síntese química , Anidridos/química , Estrutura Molecular , Fosfatos/química
7.
J Proteome Res ; 12(9): 4018-27, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23937658

RESUMO

Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein-protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/isolamento & purificação , Mapeamento de Interação de Proteínas/métodos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Proteína alfa Estimuladora de Ligação a CCAAT/química , Linhagem Celular , Cromatografia de Afinidade , Cromatografia de Fase Reversa , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/isolamento & purificação , Ligação Proteica , Mapas de Interação de Proteínas , Ratos , Estreptavidina/biossíntese , Estreptavidina/química , Estreptavidina/isolamento & purificação
9.
Cell Rep ; 42(9): 113120, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37703175

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.


Assuntos
Proteínas de Homeodomínio , Distrofia Muscular Facioescapuloumeral , Humanos , Esclerose Lateral Amiotrófica/genética , Regulação da Expressão Gênica , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Doenças Neurodegenerativas/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo
10.
Sci Adv ; 9(16): eadf5330, 2023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-37075125

RESUMO

Mixed-lineage leukemia 1 (MLL1) is a transcription activator of the HOX family, which binds to specific epigenetic marks on histone H3 through its third plant homeodomain (PHD3) domain. Through an unknown mechanism, MLL1 activity is repressed by cyclophilin 33 (Cyp33), which binds to MLL1 PHD3. We determined solution structures of Cyp33 RNA recognition motif (RRM) free, bound to RNA, to MLL1 PHD3, and to both MLL1 and the histone H3 lysine N6-trimethylated. We found that a conserved α helix, amino-terminal to the RRM domain, adopts three different positions facilitating a cascade of binding events. These conformational changes are triggered by Cyp33 RNA binding and ultimately lead to MLL1 release from the histone mark. Together, our mechanistic findings rationalize how Cyp33 binding to MLL1 can switch chromatin to a transcriptional repressive state triggered by RNA binding as a negative feedback loop.


Assuntos
Histonas , Leucemia , Humanos , Histonas/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA
11.
Mol Ther Nucleic Acids ; 34: 102052, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38028201

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2 UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

13.
Front Mol Biosci ; 9: 1062448, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36452457

RESUMO

Proximity ligation technologies are extremely powerful tools for unveiling RNA-protein interactions occurring at different stages in living cells. These approaches mainly rely on the inducible activity of enzymes (biotin ligases or peroxidases) that promiscuously biotinylate macromolecules within a 20 nm range. These enzymes can be either fused to an RNA binding protein or tethered to any RNA of interest and expressed in living cells to biotinylate the amino acids and nucleic acids of binding partners in proximity. The biotinylated molecules can then be easily affinity purified under denaturing conditions and analyzed by mass spectrometry or next generation sequencing. These approaches have been widely used in recent years, providing a potent instrument to map the molecular interactions of specific RNA-binding proteins as well as RNA transcripts occurring in mammalian cells. In addition, they permit the identification of transient interactions as well as interactions among low expressed molecules that are often missed by standard affinity purification strategies. This review will provide a brief overview of the currently available proximity ligation methods, highlighting both their strengths and shortcomings. Furthermore, it will bring further insights to the way these technologies could be further used to characterize post-transcriptional modifications that are known to regulate RNA-protein interactions.

14.
Mol Cell Oncol ; 7(4): 1743808, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32944613

RESUMO

We have recently shown that arginine methylation by protein arginine N-methyltransferase 1 (PRMT1) controls the response to cisplatin in ovarian cancer cells. In addition to increased methylation of chromatin proteins that favors senescence-associated secretory phenotype (SASP) activation, our study unraveled global hypo-methylation of RNA-binding proteins, which - we speculate - may promote their phase separation and stress granules formation.

15.
Cell Rep ; 30(4): 1208-1222.e9, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995759

RESUMO

Protein arginine methyltransferase 1 (PRMT1) is overexpressed in various human cancers and linked to poor response to chemotherapy. Various PRMT1 inhibitors are currently under development; yet, we do not fully understand the mechanisms underpinning PRMT1 involvement in tumorigenesis and chemoresistance. Using mass spectrometry-based proteomics, we identified PRMT1 as regulator of arginine methylation in ovarian cancer cells treated with cisplatin. We showed that DNA-dependent protein kinase (DNA-PK) binds to and phosphorylates PRMT1 in response to cisplatin, inducing its chromatin recruitment and redirecting its enzymatic activity toward Arg3 of histone H4 (H4R3). On chromatin, the DNA-PK/PRMT1 axis induces senescence-associated secretory phenotype through H4R3me2a deposition at pro-inflammatory gene promoters. Finally, PRMT1 inhibition reduces the clonogenic growth of cancer cells exposed to low doses of cisplatin, sensitizing them to apoptosis. While unravelling the role of PRMT1 in response to genotoxic agents, our findings indicate the possibility of targeting PRMT1 to overcome chemoresistance in cancer.


Assuntos
Antineoplásicos/farmacologia , Senescência Celular/efeitos dos fármacos , Cromatina/metabolismo , Cisplatino/farmacologia , Proteína Quinase Ativada por DNA/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Arginina/metabolismo , Senescência Celular/genética , Imunoprecipitação da Cromatina , Cromatografia Líquida , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Espectrometria de Massas , Metilação , NF-kappa B/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteoma/química , Proteoma/metabolismo , RNA-Seq , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Espectrometria de Massas em Tandem
16.
Front Mol Biosci ; 5: 90, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30467545

RESUMO

The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets.

18.
Sci Rep ; 6: 28107, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27346722

RESUMO

Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[(18)O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Proteínas do Citoesqueleto/metabolismo , Espectrometria de Massas , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , RNA Helicases DEAD-box/química , Células HEK293 , Humanos , Cinética , Isótopos de Oxigênio/química , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Especificidade por Substrato
19.
Nat Genet ; 46(9): 1021-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25129144

RESUMO

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.


Assuntos
Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Neutropenia/congênito , Adolescente , Adulto , Apoptose/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Glicosilação , Homeostase/genética , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/metabolismo , Mutação , Neutropenia/genética , Neutropenia/metabolismo , Neutropenia/patologia , Neutrófilos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais , Adulto Jovem
20.
Genome Biol ; 14(7): R81, 2013 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-23902751

RESUMO

BACKGROUND: The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported. RESULTS: We designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function. CONCLUSIONS: The dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.


Assuntos
Ácidos Nucleicos/metabolismo , Mapeamento de Interação de Proteínas , Sequência de Bases , Linhagem Celular , Bases de Dados de Proteínas , Doença , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Especificidade por Substrato
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