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Akey et al.1 use complementary experimental approaches and AI-based structure prediction to reveal new details of the structure of the yeast nuclear pore complex, providing key insights into evolution, assembly, and nucleocytoplasmic transport mechanisms.
Assuntos
Bolsas de Estudo , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Poro NuclearRESUMO
In this issue of Molecular Cell, Roake et al. (2019) define a feedforward kinetic pathway consisting of a cycle of oligoadenylation and deadenylation that regulates the production of mature human telomerase RNA.
Assuntos
Telomerase , Humanos , Cinética , RNARESUMO
The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.
Assuntos
Exossomos , RNA , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Exossomos/metabolismo , Proteômica , Estruturas R-Loop , RNA/metabolismo , RNA Helicases/metabolismo , RNA Nuclear/metabolismo , Linhagem Celular , Animais , CamundongosRESUMO
The RNA exosome is an evolutionarily conserved complex required for both precise RNA processing and decay. Pathogenic variants in EXOSC genes, which encode structural subunits of this complex, are linked to several autosomal recessive disorders. Here, we describe a missense allele of the EXOSC4 gene that causes a collection of clinical features in two affected siblings. This missense variant (NM_019037.3: exon3:c.560T>C) changes a leucine residue within a conserved region of EXOSC4 to proline (p.Leu187Pro). The two affected individuals show prenatal growth restriction, failure to thrive, global developmental delay, intracerebral and basal ganglia calcifications, and kidney failure. Homozygosity for the damaging variant was identified by exome sequencing with Sanger sequencing to confirm segregation. To explore the functional consequences of this amino acid change, we modeled EXOSC4-L187P in the corresponding budding yeast protein, Rrp41 (Rrp41-L187P). Cells that express Rrp41-L187P as the sole copy of the essential Rrp41 protein show growth defects. Steady-state levels of both Rrp41-L187P and EXOSC4-L187P are decreased compared to controls, and EXOSC4-L187P shows decreased copurification with other RNA exosome subunits. RNA exosome target transcripts accumulate in rrp41-L187P cells, including the 7S precursor of 5.8S rRNA. Polysome profiles show a decrease in actively translating ribosomes in rrp41-L187P cells as compared to control cells with the incorporation of 7S pre-rRNA into polysomes. This work adds EXOSC4 to the structural subunits of the RNA exosome that have been linked to human disease and defines foundational molecular defects that could contribute to the adverse phenotypes caused by EXOSC pathogenic variants.
RESUMO
RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
Assuntos
Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Mutação de Sentido Incorreto , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Nanismo/enzimologia , Nanismo/genética , Nanismo/patologia , Exorribonucleases/química , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Fácies , Expressão Gênica , Glicina/química , Glicina/metabolismo , Perda Auditiva/enzimologia , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , RNA Fúngico/química , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , SíndromeRESUMO
The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.
Assuntos
Doenças Cerebelares/genética , Proteínas do Citoesqueleto/genética , Drosophila melanogaster/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Substituição de Aminoácidos/genética , Animais , Sistemas CRISPR-Cas/genética , Doenças Cerebelares/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Exossomos/genética , Humanos , Mutação/genética , Neurônios/patologia , RNA/genéticaRESUMO
The importin α/ß transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin-ß, by the importin-α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin-α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin-α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin-α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N-terminus are required for both Nup2 interaction with importin-α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS-cargo release in the nucleus.
Assuntos
Sinais de Localização Nuclear , Proteínas de Saccharomyces cerevisiae , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Sinais de Localização Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMO
In 2020, the American Society of Biochemistry and Molecular Biology (ASBMB) Women in Biochemistry and Molecular Biology Committee introduced the ASBMB Leadership Awards to recognize individuals with a strong commitment to advancing the careers of women in biochemistry and molecular biology along with demonstrated excellence in research, discovery, and/or service. This innovative award recognizes efforts to mentor and support trainees and colleagues at all levels. Such a leadership award provides the opportunity to focus briefly on the important role of mentoring within the STEM disciplines. The goal of this commentary, which brings together perspectives from a senior scientist and recent recipient of the ASBMB Mid-Career Leadership Award as well as two junior faculty, is to highlight approaches for purposeful support of colleagues, with an emphasis on going beyond formal mentoring committees. The commentary primarily focuses on mentoring within the academic arena of extramural funding and publication, highlighting the reality that multiple mentors with diverse expertise and perspectives are critical to support success within STEM careers.
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Tutoria/métodos , Tutoria/tendências , Mentores/psicologia , Docentes , Humanos , Pesquisadores , Sexismo/prevenção & controle , Sexismo/tendências , Estados UnidosRESUMO
The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. Here, we identify roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPase-activating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA-binding proteins in neurodevelopment.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neurogênese , Proteoma/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Contactinas/genética , Contactinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Memória , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that coimmunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA-binding protein interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RNA-binding protein with multiple roles in posttranscriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non-cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted prosurvival signals.
Assuntos
Astrócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Imunoprecipitação , Neuroproteção/fisiologia , Fosfatidilinositol 3-Quinases/química , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a RNA/genética , Espectrometria de Massas em TandemRESUMO
The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5-6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.
Assuntos
Antígenos de Neoplasias/genética , Cerebelo/anormalidades , Deficiências do Desenvolvimento/genética , Nanismo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Malformações do Sistema Nervoso/genética , Proteínas de Ligação a RNA/genética , Animais , Cerebelo/patologia , Deficiências do Desenvolvimento/patologia , Nanismo/patologia , Mutação da Fase de Leitura/genética , Homozigoto , Humanos , Mutação de Sentido Incorreto/genética , Malformações do Sistema Nervoso/patologia , Linhagem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.
Assuntos
Alanina/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Oculofaríngea/metabolismo , Proteínas Nucleares/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Proteômica , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eletroporação , Feminino , Masculino , Camundongos , Peso Molecular , Distrofia Muscular Oculofaríngea/genética , Proteína I de Ligação a Poli(A)/genética , Estudo de Prova de Conceito , Ligação ProteicaRESUMO
Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated in vitro, revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers ex vivo confirmed differences in nuclear import rates among myonuclei. Analyzing nuclear import throughout myogenesis revealed that cNLS and non-cNLS import varies during differentiation. Taken together, our results suggest that both spatial and temporal regulation of nuclear import pathways are important in muscle cell differentiation and protein regionalization in myofibers.
Assuntos
Núcleo Celular/metabolismo , Células Musculares/citologia , Células Musculares/metabolismo , Músculo Esquelético/citologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Sinais de Localização Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The RNA exosome is an evolutionarily conserved, ribonuclease complex that is critical for both processing and degradation of a variety of RNAs. Cofactors that associate with the RNA exosome likely dictate substrate specificity for this complex. Recently, mutations in genes encoding both structural subunits of the RNA exosome and its cofactors have been linked to human disease. Mutations in the RNA exosome genes EXOSC3 and EXOSC8 cause pontocerebellar hypoplasia type 1b (PCH1b) and type 1c (PCH1c), respectively, which are similar autosomal-recessive, neurodegenerative diseases. Mutations in the RNA exosome gene EXOSC2 cause a distinct syndrome with various tissue-specific phenotypes including retinitis pigmentosa and mild intellectual disability. Mutations in genes that encode RNA exosome cofactors also cause tissue-specific diseases with complex phenotypes. How mutations in these genes give rise to distinct, tissue-specific diseases is not clear. In this review, we discuss the role of the RNA exosome complex and its cofactors in human disease, consider the amino acid changes that have been implicated in disease, and speculate on the mechanisms by which exosome gene mutations could underlie dysfunction and disease.
Assuntos
Doença/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Mutação , Coenzimas/genética , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Humanos , Subunidades Proteicas/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Histone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs. While H2B ubiquitylation controls the recruitment of the different Mex67 adaptors to mRNPs, the effect of Swd2 ubiquitylation is restricted to Yra1 and Nab2, which, in turn, controls poly(A) tail length. Modification of H2B thus participates in the crosstalk between cotranscriptional events and assembly of mRNPs linking nuclear processing and mRNA export.
Assuntos
Histonas/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitinação , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The polyadenosine RNA-binding protein ZC3H14 is important in RNA processing. Although ZC3H14 is ubiquitously expressed, mutation of the ZC3H14 gene causes a non-syndromic form of intellectual disability. Here, we examine the function of ZC3H14 in the brain by identifying ZC3H14-interacting proteins using unbiased mass spectrometry. Through this analysis, we identified physical interactions between ZC3H14 and multiple RNA processing factors. Notably, proteins that comprise the THO complex were amongst the most enriched proteins. We demonstrate that ZC3H14 physically interacts with THO components and that these proteins are required for proper RNA processing, as loss of ZC3H14 or THO components leads to extended bulk poly(A) tail length. Furthermore, we identified the transcripts Atp5g1 and Psd95 as shared RNA targets of ZC3H14 and the THO complex. Our data suggest that ZC3H14 and the THO complex are important for proper processing of Atp5g1 and Psd95 RNA, as depletion of ZC3H14 or THO components leads to decreased steady-state levels of each mature transcript accompanied by accumulation of Atp5g1 and Psd95 pre-mRNA in the cytoplasm. Taken together, this work provides the first unbiased identification of nuclear ZC3H14-interacting proteins from the brain and links the functions of ZC3H14 and the THO complex in the processing of RNA.
Assuntos
Encéfalo/metabolismo , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Poli A/metabolismo , Proteínas de Ligação a Poli(A)RESUMO
RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the Pabpn1 transcript is unstable, contributing to low steady-state Pabpn1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate Pabpn1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate Pabpn1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases Pabpn1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates Pabpn1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.
Assuntos
Proteína Semelhante a ELAV 1/genética , Regulação da Expressão Gênica , Proteína I de Ligação a Poli(A)/genética , Proteínas de Ligação a RNA/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Mutação , Células NIH 3T3 , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.
Assuntos
Transformação Celular Neoplásica , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Instabilidade Genômica , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Dano ao DNA , Replicação do DNA , Desoxirribonuclease (Dímero de Pirimidina)/genética , Células Epiteliais/enzimologia , Humanos , Neoplasias Pulmonares/enzimologia , Mutação , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologiaRESUMO
Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice.
Assuntos
Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Proteína I de Ligação a Poli(A)/genética , Proteína I de Ligação a Poli(A)/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Genótipo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Peptídeos , FenótipoRESUMO
A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.