Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.562
Filtrar
1.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
2.
Nat Commun ; 11(1): 4090, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796831

RESUMO

hnRNPA2 is a human ribonucleoprotein (RNP) involved in RNA metabolism. It forms fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and missense mutations in this domain are found in the disease multisystem proteinopathy (MSP). However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of the hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into ß-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment (D290V) of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to a pathogenic form.


Assuntos
Amiloide/química , Amiloide/metabolismo , Microscopia Crioeletrônica/métodos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/ultraestrutura , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Humanos , Hidrogéis/química , Proteínas de Ligação a RNA/química
3.
Nat Commun ; 11(1): 3751, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719344

RESUMO

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.


Assuntos
Ribonucleoproteínas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Rotação , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação/genética , Domínios Proteicos , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Subunidades Ribossômicas Maiores/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
4.
Nucleic Acids Res ; 48(13): 7421-7438, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496517

RESUMO

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Quadruplex G , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Camundongos , Ligação Proteica , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química
5.
Nucleic Acids Res ; 48(12): 6491-6502, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484544

RESUMO

Multifunctional proteins often perform their different functions when localized in different subcellular compartments. However, the mechanisms leading to their localization are largely unknown. Recently, 3'UTRs were found to regulate the cellular localization of newly synthesized proteins through the formation of 3'UTR-protein complexes. Here, we investigate the formation of 3'UTR-protein complexes involving multifunctional proteins by exploiting large-scale protein-protein and protein-RNA interaction networks. Focusing on 238 human 'extreme multifunctional' (EMF) proteins, we predicted 1411 3'UTR-protein complexes involving 54% of those proteins and evaluated their role in regulating protein cellular localization and multifunctionality. We find that EMF proteins lacking localization addressing signals, yet present at both the nucleus and cell surface, often form 3'UTR-protein complexes, and that the formation of these complexes could provide EMF proteins with the diversity of interaction partners necessary to their multifunctionality. Our findings are reinforced by archetypal moonlighting proteins predicted to form 3'UTR-protein complexes. Finally, the formation of 3'UTR-protein complexes that involves up to 17% of the proteins in the human protein-protein interaction network, may be a common and yet underestimated protein trafficking mechanism, particularly suited to regulate the localization of multifunctional proteins.


Assuntos
Regiões 3' não Traduzidas , Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos , Proteínas de Membrana/química , Ligação Proteica , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas , Transporte Proteico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química
6.
Nucleic Acids Res ; 48(13): 7385-7403, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32491174

RESUMO

Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.


Assuntos
Elementos Ricos em Adenilato e Uridilato , Proteínas de Ligação a RNA/química , Ubiquitina-Proteína Ligases/química , Sítios de Ligação , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Motivos de Nucleotídeos , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
Nucleic Acids Res ; 48(13): 7502-7519, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542384

RESUMO

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3'-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least 2 bp preceding an 3' oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5' side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3' and cspE-3' RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3' intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Ligação a RNA/química , Sítios de Ligação , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/química , Fator Proteico 1 do Hospedeiro/metabolismo , Mutação , Motivos de Nucleotídeos , Ligação Proteica , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
8.
Nucleic Acids Res ; 48(14): 8006-8021, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32556302

RESUMO

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.


Assuntos
Iniciação Traducional da Cadeia Peptídica , Poliovirus/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
9.
Nucleic Acids Res ; 48(14): 7958-7972, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32597966

RESUMO

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.


Assuntos
Adenosina Desaminase/química , Adenosina Desaminase/metabolismo , Edição de RNA , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA de Cadeia Dupla/química , Proteínas de Ligação a RNA/genética
10.
Nat Commun ; 11(1): 2823, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499480

RESUMO

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Neisseria meningitidis/genética , Conformação de Ácido Nucleico , Estresse Oxidativo , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
11.
Nucleic Acids Res ; 48(10): 5235-5253, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356888

RESUMO

Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.


Assuntos
Oligonucleotídeos Fosforotioatos/química , Proteínas/química , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ligantes , Oligonucleotídeos Fosforotioatos/metabolismo , Oligonucleotídeos Fosforotioatos/farmacologia , Oligonucleotídeos Fosforotioatos/toxicidade , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteínas/toxicidade , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
12.
Nucleic Acids Res ; 48(11): 5859-5872, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421779

RESUMO

Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization.


Assuntos
Fracionamento Celular , Microambiente Celular , Espaço Intracelular/química , RNA/análise , RNA/metabolismo , Análise de Sequência de RNA , Transcriptoma , Animais , Extratos Celulares/química , Centrifugação com Gradiente de Concentração , Feminino , Fígado/citologia , Fígado/metabolismo , Espectrometria de Massas , Camundongos , RNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Sacarose
13.
Mol Cell ; 78(1): 9-29, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32243832

RESUMO

RNA-binding proteins (RBPs) comprise a large class of over 2,000 proteins that interact with transcripts in all manner of RNA-driven processes. The structures and mechanisms that RBPs use to bind and regulate RNA are incredibly diverse. In this review, we take a look at the components of protein-RNA interaction, from the molecular level to multi-component interaction. We first summarize what is known about protein-RNA molecular interactions based on analyses of solved structures. We additionally describe software currently available for predicting protein-RNA interaction and other resources useful for the study of RBPs. We then review the structure and function of seventeen known RNA-binding domains and analyze the hydrogen bonds adopted by protein-RNA structures on a domain-by-domain basis. We conclude with a summary of the higher-level mechanisms that regulate protein-RNA interactions.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Motivos de Ligação ao RNA , Software
14.
Nucleic Acids Res ; 48(10): 5670-5683, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329775

RESUMO

Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.


Assuntos
Ciclofilinas/química , Fator de Iniciação 4A em Eucariotos/química , Proteínas de Ligação a RNA/química , Spliceossomos/química , Linhagem Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Inflamação/genética , Modelos Moleculares , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Spliceossomos/metabolismo
15.
Nat Struct Mol Biol ; 27(4): 363-372, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231288

RESUMO

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.


Assuntos
Proteínas de Choque Térmico HSP27/química , Chaperonas Moleculares/química , Proteína FUS de Ligação a RNA/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/isolamento & purificação , Humanos , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Fosforilação , Ligação Proteica/genética , Domínios Proteicos/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Estresse Fisiológico/genética
16.
PLoS One ; 15(4): e0231114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32243476

RESUMO

Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Fusão Celular , Sequência Conservada , Ciclina B , Regulação para Baixo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica , Meiose , Proteínas Mutantes/metabolismo , Mutação/genética , Oogênese , Ovário/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Tiorredoxinas/metabolismo
17.
Nucleic Acids Res ; 48(9): 4827-4838, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32286661

RESUMO

NONO is a DNA/RNA-binding protein, which plays a critical regulatory role during cell stage transitions of mouse embryonic stem cells (mESCs). However, its function in neuronal lineage commitment and the molecular mechanisms of its action in such processes are largely unknown. Here we report that NONO plays a key role during neuronal differentiation of mESCs. Nono deletion impedes neuronal lineage commitment largely due to a failure of up-regulation of specific genes critical for neuronal differentiation. Many of the NONO regulated genes are also DNA demethylase TET1 targeted genes. Importantly, re-introducing wild type NONO to the Nono KO cells, not only restores the normal expression of the majority of NONO/TET1 coregulated genes but also rescues the defective neuronal differentiation of Nono-deficient mESCs. Mechanistically, our data shows that NONO directly interacts with TET1 via its DNA binding domain and recruits TET1 to genomic loci to regulate 5-hydroxymethylcytosine levels. Nono deletion leads to a significant dissociation of TET1 from chromatin and dysregulation of DNA hydroxymethylation of neuronal genes. Taken together, our findings reveal a key role and an epigenetic mechanism of action of NONO in regulation of TET1-targeted neuronal genes, offering new functional and mechanistic understanding of NONO in stem cell functions, lineage commitment and specification.


Assuntos
Cromatina/enzimologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Neurogênese/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/fisiologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Camundongos , Proteínas Proto-Oncogênicas/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Transcrição Genética
18.
Nat Commun ; 11(1): 1926, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317642

RESUMO

MicroRNAs (miRNAs) are small RNAs that regulate gene expression. miRNAs are produced from primary miRNAs (pri-miRNAs), which are cleaved by Microprocessor. Microprocessor, therefore, plays a crucial role in determining the efficiency and precision of miRNA production, and thus the function of the final miRNA product. Here, we conducted high-throughput enzymatic assays to investigate the catalytic mechanism of Microprocessor cleaving randomized pri-miRNAs. We identified multiple mismatches and wobble base pairs in the upper stem of pri-miRNAs, which influence the efficiency and accuracy of their processing. The existence of these RNA elements helps to explain the alternative cleavage of Microprocessor for some human pri-miRNAs. We also demonstrated that miRNA biogenesis can be altered via modification of the RNA elements by RNA-editing events or single nucleotide polymorphisms (SNPs). These findings improve our understanding of pri-miRNA processing mechanisms and provide a foundation for interpreting differential miRNA expression due to RNA modifications and SNPs.


Assuntos
MicroRNAs/metabolismo , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Pareamento de Bases , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Edição de RNA , Interferência de RNA , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Ribonuclease III/metabolismo , Análise de Sequência de RNA
19.
Proc Natl Acad Sci U S A ; 117(15): 8503-8514, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234784

RESUMO

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/química
20.
PLoS One ; 15(4): e0232366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348368

RESUMO

Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P (canonical length) and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.


Assuntos
Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Edição de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Bryopsida/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/química , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA