RESUMO
Membraneless organelles (MLOs) are detected in cells as dots of mesoscopic size. By undergoing phase separation into a liquid-like or gel-like phase, MLOs contribute to intracellular compartmentalization of specific biological functions. In eukaryotes, dozens of MLOs have been identified, including the nucleolus, Cajal bodies, nuclear speckles, paraspeckles, promyelocytic leukaemia protein (PML) nuclear bodies, nuclear stress bodies, processing bodies (P bodies) and stress granules. MLOs contain specific proteins, of which many possess intrinsically disordered regions (IDRs), and nucleic acids, mainly RNA. Many MLOs contribute to gene regulation by different mechanisms. Through sequestration of specific factors, MLOs promote biochemical reactions by simultaneously concentrating substrates and enzymes, and/or suppressing the activity of the sequestered factors elsewhere in the cell. Other MLOs construct inter-chromosomal hubs by associating with multiple loci, thereby contributing to the biogenesis of macromolecular machineries essential for gene expression, such as ribosomes and spliceosomes. The organization of many MLOs includes layers, which might have different biophysical properties and functions. MLOs are functionally interconnected and are involved in various diseases, prompting the emergence of therapeutics targeting them. In this Review, we introduce MLOs that are relevant to gene regulation and discuss their assembly, internal structure, gene-regulatory roles in transcription, RNA processing and translation, particularly in stress conditions, and their disease relevance.
Assuntos
Condensados Biomoleculares , Organelas , Organelas/metabolismo , RNA/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismoRESUMO
Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress-inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature-dependent Ser/Arg-rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA-binding proteins revealed HSATIII association with proteins related to N6 -methyladenosine (m6 A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m6 A-modified. nSBs sequester the m6 A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m6 A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m6 A modification of pre-mRNAs, leading to repression of m6 A-dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature-dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m6 A-modified architectural lncRNAs.
Assuntos
Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , TemperaturaRESUMO
Nuclear stress bodies (nSBs) are thermal stress-inducible membrane-less nuclear bodies that are formed on highly repetitive satellite III architectural noncoding RNAs (HSATIII arcRNAs). Upon thermal stress exposure, HSATIII expression is induced to sequestrate specific sets of RNA-binding proteins and form nSBs. The major population of nSBs contain SAFB as a marker, whereas the minor population are SAFB-negative. Here, we found that HNRNPM, which was previously reported to localize in nuclear foci adjacent to SAFB-positive foci upon thermal stress, localizes in a minor population of HSATIII-dependent nSBs. Hence, we used the terms nSB-S and nSB-M to distinguish the SAFB foci and HNRNPM foci, respectively. Analysis of the components of the nSBs revealed that each set contains distinct RNA-binding proteins, including SLTM and NCO5A in nSB-Ss and HNRNPA1 and HNRNPH1 in nSB-Ms. Overall, our findings indicate that two sets of nSBs containing HSATIII arcRNAs and distinct sets of RNA-binding proteins are formed upon thermal stress exposure.
Assuntos
Núcleo Celular/metabolismo , RNA Satélite/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Estresse Fisiológico , Temperatura , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases.
Assuntos
Encéfalo , Transtornos Mentais , Doenças Neurodegenerativas , Neurônios/metabolismo , Fator de Processamento Associado a PTB/deficiência , Elongação da Transcrição Genética , Animais , Apoptose , Encéfalo/metabolismo , Encéfalo/patologia , Quinase 9 Dependente de Ciclina/metabolismo , Íntrons , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Transtornos Mentais/patologia , Camundongos , Camundongos Knockout , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Fator de Processamento Associado a PTB/metabolismo , RNA Polimerase II/metabolismoRESUMO
The 5' splice site mutation (IVS20+6T>C) of the inhibitor of κ light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene in familial dysautonomia (FD) is at the sixth intronic nucleotide of the 5' splice site. It is known to weaken U1 snRNP recognition and result in an aberrantly spliced mRNA product in neuronal tissue, but normally spliced mRNA in other tissues. Aberrantly spliced IKBKAP mRNA abrogates IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1) expression and results in a defect of neuronal cell development in FD. To elucidate the tissue-dependent regulatory mechanism, we screened an expression library of major RNA-binding proteins (RBPs) with our mammalian dual-color splicing reporter system and identified RBM24 as a regulator. RBM24 functioned as a cryptic intronic splicing enhancer binding to an element (IVS20+13-29) downstream from the intronic 5' splice site mutation in the IKBKAP gene and promoted U1 snRNP recognition only to the mutated 5' splice site (and not the wild-type 5' splice site). Our results show that tissue-specific expression of RBM24 can explain the neuron-specific aberrant splicing of IKBKAP exon 20 in familial dysautonomia, and that ectopic expression of RBM24 in neuronal tissue could be a novel therapeutic target of the disease.
Assuntos
Proteínas de Transporte/genética , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Mutação , Sítios de Splice de RNA , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Processamento Alternativo , Animais , Linhagem Celular , Éxons , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Íntrons , Camundongos , Modelos Biológicos , Especificidade de Órgãos/genética , Ligação Proteica , Precursores de RNA/química , Precursores de RNA/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Elongação da TranscriçãoRESUMO
The abnormal expression and function of myelin-related proteins contribute to nervous system dysfunction associated with neuropsychiatric disorders; however, the underlying mechanism of this remains unclear. We found here that breast carcinoma amplified sequence 1 (BCAS1), a basic protein abundant in the brain, was expressed specifically in oligodendrocytes and Schwann cells, and that its expression level was decreased by demyelination. This suggests that BCAS1 is a novel myelin-associated protein. BCAS1 knockout mice displayed schizophrenia-like behavioral abnormalities and a tendency toward reduced anxiety-like behaviors. Moreover, we found that the loss of BCAS1 specifically induced hypomyelination and the expression of inflammation-related genes in the brain. These observations provide a novel insight into the functional link between oligodendrocytes and inflammation and/or abnormal behaviors.
Assuntos
Encéfalo/metabolismo , Doenças Desmielinizantes/genética , Proteínas de Neoplasias/metabolismo , Oligodendroglia/metabolismo , Animais , Encéfalo/patologia , Doenças Desmielinizantes/patologia , Inflamação/genética , Camundongos Knockout , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas de Neoplasias/deficiência , Esquizofrenia/genética , Esquizofrenia/patologia , Ativação Transcricional/genética , Regulação para CimaRESUMO
Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Neovascularização de Coroide/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Pirimidinonas/administração & dosagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , Tiadiazóis/administração & dosagem , Administração Tópica , Animais , Linhagem Celular , Cristalografia por Raios X , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologia , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Niacinamida/análogos & derivados , Niacinamida/química , Piperidinas/química , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinonas/química , Pirimidinonas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tiadiazóis/química , Tiadiazóis/farmacologiaRESUMO
1. PMES-2 is a protein, of which mRNA is translocated to the neurites of hippocampal neurons. Since the protein is present in the postsynaptic density, contributions to synaptic function have been predicted. 2. To elucidate the protein-protein interaction of PMES-2, yeast two-hybrid screening was performed with PMES-2 partial polypeptides as baits. We found that PMES-2 interacted with dynein light chain-2 (DLC-2), a light chain subunit of myosin-V and cytoplasmic dynein, via the C-terminal 20 amino acids. Exogenous PMES-2 colocalized with F-actin at the cell periphery, while a PMES-2 mutant lacking the DLC-binding site localized primarily in the nucleus. 3. This dual-targeting of PMES-2 constructs depends on an effector domain-like motif in the N-terminus. 4. These results indicate that PMES-2 links a motor complex to the membrane skeleton and that DLC-1/2 inhibits PMES-2 nuclear localization. PMES-2 possibly modifies the cytoskeletal architecture and protein transport at the synapse and/or regulates signal transduction from the synapse to the nucleus.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Células Cultivadas , Citoplasma/metabolismo , Proteína GAP-43/química , Proteína GAP-43/genética , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Neurônios/ultraestrutura , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Although a subpopulation of mRNAs has been identified as translocated to the dendrites or the synaptic regions of neurons, the translocational mechanism has not been elucidated. To find mRNAs enriched in synapses, we compared the synaptosomal mRNAs with those from whole forebrain using differential display (DD). We cloned one of these mRNAs, which encoded a novel 31 kDa protein (PMES-2). PMES-2 mRNA was specifically transcribed in the brain and was present in the dendrites of the hippocampal neurons. PMES-2 protein was partly localized in the postsynaptic density. Although this protein is very similar to human NABC1 protein, its function is still unknown.