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1.
Genes Dev ; 36(3-4): 180-194, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35058317

RESUMO

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.


Assuntos
Meiose , RNA Helicases , Animais , Regulação da Expressão Gênica , Masculino , Mamíferos/genética , Meiose/genética , Camundongos , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese/genética
2.
Proc Natl Acad Sci U S A ; 121(10): e2314695121, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38416679

RESUMO

NOVA1 is a neuronal RNA-binding protein identified as the target antigen of a rare autoimmune disorder associated with cancer and neurological symptoms, termed paraneoplastic opsoclonus-myoclonus ataxia. Despite the strong association between NOVA1 and cancer, it has been unclear how NOVA1 function might contribute to cancer biology. In this study, we find that NOVA1 acts as an oncogenic factor in a GBM (glioblastoma multiforme) cell line established from a patient. Interestingly, NOVA1 and Argonaute (AGO) CLIP identified common 3' untranslated region (UTR) targets, which were down-regulated in NOVA1 knockdown GBM cells, indicating a transcriptome-wide intersection of NOVA1 and AGO-microRNA (miRNA) targets regulation. NOVA1 binding to 3'UTR targets stabilized transcripts including those encoding cholesterol homeostasis related proteins. Selective inhibition of NOVA1-RNA interactions with antisense oligonucleotides disrupted GBM cancer cell fitness. The precision of our GBM CLIP studies point to both mechanism and precise RNA sequence sites to selectively inhibit oncogenic NOVA1-RNA interactions. Taken together, we find that NOVA1 is commonly overexpressed in GBM, where it can antagonize AGO2-miRNA actions and consequently up-regulates cholesterol synthesis, promoting cell viability.


Assuntos
Glioblastoma , MicroRNAs , Humanos , Glioblastoma/genética , Glioblastoma/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , MicroRNAs/genética , Homeostase/genética , Colesterol , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Antígeno Neuro-Oncológico Ventral
3.
Genes Dev ; 31(10): 990-1006, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28637692

RESUMO

Understanding the biologic role of N6-methyladenosine (m6A) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and m6A in exons but very rarely in introns. The m6A methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each m6A modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of m6As in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring m6A in wild-type mouse stem cells is spliced the same in cells lacking the major m6A methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring m6As have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, m6A is added to exons before or soon after exon definition in nascent pre-mRNA, and while m6A is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.


Assuntos
Citoplasma/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Animais , Cromatina/metabolismo , Células-Tronco Embrionárias , Éxons/genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Íntrons/genética , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos
4.
N Engl J Med ; 384(23): 2212-2218, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33882219

RESUMO

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of clinical concern. In a cohort of 417 persons who had received the second dose of BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccine at least 2 weeks previously, we identified 2 women with vaccine breakthrough infection. Despite evidence of vaccine efficacy in both women, symptoms of coronavirus disease 2019 developed, and they tested positive for SARS-CoV-2 by polymerase-chain-reaction testing. Viral sequencing revealed variants of likely clinical importance, including E484K in 1 woman and three mutations (T95I, del142-144, and D614G) in both. These observations indicate a potential risk of illness after successful vaccination and subsequent infection with variant virus, and they provide support for continued efforts to prevent and diagnose infection and to characterize variants in vaccinated persons. (Funded by the National Institutes of Health and others.).


Assuntos
Anticorpos Neutralizantes/sangue , Vacinas contra COVID-19 , COVID-19/virologia , Mutação , SARS-CoV-2/genética , Vacina de mRNA-1273 contra 2019-nCoV , Idoso , Anticorpos Antivirais/sangue , Vacina BNT162 , COVID-19/diagnóstico , Feminino , Humanos , Pessoa de Meia-Idade , Testes de Neutralização , Filogenia , Reação em Cadeia da Polimerase , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/genética , Carga Viral
5.
Nucleic Acids Res ; 49(12): 6849-6862, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34157123

RESUMO

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establishes a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circEfnb2 was impaired when YCAY clusters located in flanking introns were mutagenized. CLIP (cross-linking and immunoprecipitation) and additional reporter analyses demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain.


Assuntos
Antígenos de Neoplasias/fisiologia , Encéfalo/metabolismo , Neurônios/metabolismo , RNA Circular/metabolismo , Proteínas de Ligação a RNA/fisiologia , Processamento Alternativo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Encéfalo/embriologia , Efrina-B2/genética , Éxons , Regulação da Expressão Gênica , Células HEK293 , Humanos , Íntrons , Camundongos Knockout , Antígeno Neuro-Oncológico Ventral , Motivos de Nucleotídeos , RNA Circular/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
6.
J Biol Chem ; 295(28): 9650-9662, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32467230

RESUMO

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic ß-amyloid (Aß) peptide generated from proteolytic cleavage of ß-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin (PSEN) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aß generation in vitro, and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism in vivo remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP in vivo We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic ß-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aß in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aß-dependent pathogenicity in AD.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Complexos Multiproteicos/metabolismo , Processamento de Proteína Pós-Traducional , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo
7.
J Neurochem ; 148(4): 480-498, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30411795

RESUMO

X11/Mint 1 and X11-like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11-, X11L-, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non-PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro-localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild-type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo
8.
Proc Natl Acad Sci U S A ; 111(7): 2638-43, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24497505

RESUMO

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß). The genes that govern this process, however, have remained elusive. To this end, we combined distinct mouse strains with transcriptomics to directly identify disease-relevant genes. We show that AD model mice (APP-Tg) with DBA/2 genetic backgrounds have significantly lower levels of Aß accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress Aß accumulation. To avoid detecting secondarily affected genes by Aß, we used non-Tg mice in the absence of Aß pathology and selected candidate genes differently expressed in DBA/2 mice. Additional transcriptome analysis of APP-Tg mice with mixed genetic backgrounds revealed kinesin light chain-1 (Klc1) as an Aß modifier, indicating a role for intracellular trafficking in Aß accumulation. Aß levels correlated with the expression levels of Klc1 splice variant E and the genotype of Klc1 in these APP-Tg mice. In humans, the expression levels of KLC1 variant E in brain and lymphocyte were significantly higher in AD patients compared with unaffected individuals. Finally, functional analysis using neuroblastoma cells showed that overexpression or knockdown of KLC1 variant E increases or decreases the production of Aß, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of Aß pathology. This unique combination of distinct mouse strains and model mice with transcriptomics is expected to be useful for the study of genetic mechanisms of other complex diseases.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Isoformas de Proteínas/metabolismo , Doença de Alzheimer/genética , Animais , Encéfalo/metabolismo , Cruzamentos Genéticos , Perfilação da Expressão Gênica , Humanos , Cinesinas , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Isoformas de Proteínas/genética , Especificidade da Espécie
9.
J Biol Chem ; 287(23): 19715-24, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22511769

RESUMO

Amyloid ß-precursor protein (APP) is primarily cleaved by α- or ß-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aß generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aß in Alzheimer disease.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Microdomínios da Membrana/metabolismo , Triptofano/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/patologia , Química Encefálica/genética , Microdomínios da Membrana/genética , Camundongos , Fosforilação , Estrutura Terciária de Proteína , Triptofano/genética
10.
Cell Rep ; 42(2): 112050, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36716149

RESUMO

We describe a patient haploinsufficient for the neuronal RNA binding protein NOVA1 who developed a behavioral motor hyperactivity disorder, suggesting a role of NOVA1 in postnatal motor inhibition. To investigate Nova1's action in adult Gad2+ inhibitory neurons, we generated a conditional Nova1-null mouse (Nova1-cKOGad2-cre). Strikingly, the phenotypes of these mice show many similarities to the NOVA1 haploinsufficient patient and identify a function of Nova1 in the hypothalamus. Molecularly, Nova1 loss in Gad2-positive neurons alters downstream expression of Impact mRNA, along with a subset of RNAs encoding electron transport chain-related factors and ribosomal proteins. NOVA1 stabilizes Impact mRNA by binding its 3' UTR, antagonizing the actions of miR-138 and miR-124. Together, these studies demonstrate actions of NOVA1 in adult hypothalamic neurons, mechanisms by which it functions in translation and metabolism, including through direct binding to Impact mRNA, and illuminate its role in human neurologic disease.


Assuntos
MicroRNAs , Antígeno Neuro-Oncológico Ventral , Proteínas de Ligação a RNA , Animais , Humanos , Camundongos , Hipotálamo/metabolismo , MicroRNAs/metabolismo , Antígeno Neuro-Oncológico Ventral/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
11.
Neurobiol Aging ; 123: 63-74, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36638682

RESUMO

Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease (AD). Here, we investigated brain amyloid-ß (Aß) accumulation throughout the aging process in an amyloid precursor protein (APP) knock-in (KI) mouse model of AD that expresses human APPNL-G-F with or without human apoE4 or apoE3. Brain Aß42 levels were significantly lower in 9-month-old mice that express human isoforms of apoE than in age-matched APP-KI control mice. Linear accumulation of Aß42 began in 5-month-old apoE4 mice, and a strong increase in Aß42 levels was observed in 21-month-old apoE3 mice. Aß42 levels in cerebroventricular fluid were higher in apoE3 than in apoE4 mice at 6-7 months of age, suggesting that apoE3 is more efficient at clearing Aß42 than apoE4 at these ages. However, apoE3 protein levels were lower than apoE4 protein levels in the brains of 21-month-old apoE3 and apoE4 mice, respectively, which may explain the rapid increase in brain Aß42 burden in apoE3 mice. We identified genes that were downregulated in a human apoE-dependent (apoE4 > apoE3) and age-dependent (apoE3 = apoE4) manner, which may regulate brain Aß burden and/or AD progression. Analysis of gene expression in AD mouse models helps identify molecular mechanisms of pleiotropy by the human APOE gene during aging.


Assuntos
Doença de Alzheimer , Humanos , Camundongos , Animais , Doença de Alzheimer/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Camundongos Transgênicos , Apolipoproteínas E/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Expressão Gênica
12.
Cell Stem Cell ; 28(5): 894-905.e7, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33631117

RESUMO

H3.3G34R-mutant gliomas are lethal tumors of the cerebral hemispheres with unknown mechanisms of regional specificity and tumorigenicity. We developed a human embryonic stem cell (hESC)-based model of H3.3G34R-mutant glioma that recapitulates the key features of the tumors with cell-type specificity to forebrain interneuronal progenitors but not hindbrain precursors. We show that H3.3G34R, ATRX, and TP53 mutations cooperatively impact alternative RNA splicing events, particularly suppression of intron retention. This leads to increased expression of components of the Notch pathway, notably NOTCH2NL, a human-specific gene family. We also uncover a parallel mechanism of enhanced NOTCH2NL expression via genomic amplification of its locus in some H3.3G34R-mutant tumors. These findings demonstrate a novel mechanism whereby evolutionary pathways that lead to larger brain size in humans are co-opted to drive tumor growth.


Assuntos
Neoplasias Encefálicas , Glioma , Células-Tronco Embrionárias Humanas , Neoplasias Encefálicas/genética , Glioma/genética , Histonas/genética , Humanos , Mutação/genética , Oncogenes
13.
J Neurochem ; 111(5): 1213-24, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19780898

RESUMO

The metabolism of amyloid beta-protein precursor (APP) is regulated by various cytoplasmic and/or membrane-associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp-His-His-Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC-12, strongly inhibited APP metabolism, including amyloid beta-protein (Abeta) generation. In cells expressing AID/DHHC-12, APP was tethered in the Golgi, and APP-containing vesicles disappeared from the cytoplasm. Although DHHC domain-containing proteins are involved in protein palmitoylation, a AID/DHHC-12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala-Ala-His-Ser (AAHS) made no detectable difference in the generation and trafficking of APP-containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC-12 significantly increased non-amyloidogenic alpha-cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major alpha-secretase, suggesting that protein palmitoylation involved in the regulation of alpha-secretase activity. AID/DHHC-12 can modify APP metabolism, including Abeta generation in multiple ways by regulating the generation and/or trafficking of APP-containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity-independent manner, and the alpha-cleavage of APP in the enzymatic activity-dependent manner.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Citidina Desaminase/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação/métodos , Camundongos , Modelos Moleculares , Mutação/genética , Fatores de Transcrição NFI/metabolismo , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico/fisiologia , Transfecção/métodos , Tubulina (Proteína)/metabolismo
14.
Neuron ; 101(4): 707-720.e5, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30638744

RESUMO

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.


Assuntos
Processamento Alternativo , Antígenos de Neoplasias/genética , Cerebelo/embriologia , Córtex Cerebral/embriologia , Neurogênese , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Cerebelo/citologia , Cerebelo/fisiologia , Córtex Cerebral/citologia , Potenciais Pós-Sinápticos Excitadores , Feminino , Potenciais Pós-Sinápticos Inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Antígeno Neuro-Oncológico Ventral , Neurônios/citologia , Neurônios/fisiologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Ligação a RNA/metabolismo
15.
Genome Biol ; 19(1): 117, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-30111345

RESUMO

BACKGROUND: Alternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell type-specific post-transcriptional regulation has been a challenge. Here, we describe a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons. RESULTS: We developed a means of undertaking motoneuron-specific CLIP to explore motoneuron-specific protein-RNA interactions relative to studies of the whole spinal cord in mouse. This allowed us to pinpoint differential RNA regulation specific to motoneurons, revealing a major role for NOVA in regulating cytoskeleton interactions in motoneurons. In particular, NOVA specifically promotes the palmitoylated isoform of the cytoskeleton protein Septin 8 in motoneurons, which enhances dendritic arborization. CONCLUSIONS: Our study demonstrates that cell type-specific RNA regulation is important for fine tuning motoneuron physiology and highlights the value of defining RNA processing regulation at single cell type resolution.


Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Citoesqueleto/metabolismo , Imunoprecipitação , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/metabolismo , Dendritos/metabolismo , Éxons/genética , Lipoilação , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Proteínas do Tecido Nervoso/química , Antígeno Neuro-Oncológico Ventral , Pseudópodes/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/química , Septinas/metabolismo , Transcriptoma/genética
16.
Neuron ; 95(6): 1334-1349.e5, 2017 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-28910620

RESUMO

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.


Assuntos
Encéfalo/citologia , Microglia/metabolismo , Neurônios/metabolismo , Poliadenilação , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antígenos de Neoplasias/fisiologia , Astrócitos/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Microglia/citologia , Proteínas do Tecido Nervoso/fisiologia , Antígeno Neuro-Oncológico Ventral , Especificidade de Órgãos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/fisiologia
17.
Brain Res ; 1646: 227-234, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27268412

RESUMO

The X11/Mint family of proteins comprises X11/X11α/Mint1, X11L/X11ß/Mint2, and X11L2/X11γ/Mint3. Each of these molecules is an adaptor protein that contains a phosphotyrosine interaction/binding (PI/PTB) and two PDZ domains in its carboxy-terminal region. X11/Mint family members associate with a broad spectrum of membrane proteins, including Alzheimer's ß-amyloid precursor protein (APP), alcadeins, and low density lipoprotein receptor proteins, as well as various cytoplasmic proteins including Arf, kalirin-7, and Munc18. In particular, X11 and X11L are thought to play various roles in the regulation of neural functions in brain. Nevertheless, the protein levels and respective localization of individual family members remain controversial. We analyzed the protein levels of X11 and X11L in the corresponding single- and double-knockout mice. X11 and X11L did not exhibit obvious changes of their protein levels when the other was absent, especially in cerebrum in which they were widely co-expressed. In cerebellum, X11 and X11L localized in characteristic patterns in various types of neurons, and X11 protein level increased without an obvious ectopic localization in X11L-knockout mice. Interestingly, only X11L protein existed specifically in brain, whereas, contrary to the accepted view, X11 protein was detected at the highest levels in brain but was also strongly detected in pancreas, testis, and paranephros. Together, our results indicate that both X11 and X11L exert largely in brain neurons, but X11 may also function in peripheral tissues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Transporte/genética , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , RNA Mensageiro/metabolismo
18.
Elife ; 52016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27223328

RESUMO

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.


Assuntos
Processamento Alternativo , Antígenos de Neoplasias/metabolismo , Orientação de Axônios , Interneurônios Comissurais/fisiologia , Receptor DCC/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Medula Espinal/embriologia , Animais , Antígenos de Neoplasias/genética , Testes Genéticos , Camundongos , Camundongos Knockout , Antígeno Neuro-Oncológico Ventral , Interferência de RNA , Proteínas de Ligação a RNA/genética
19.
Cell Rep ; 15(2): 423-35, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-27050522

RESUMO

Accurate and precise annotation of 3' UTRs is critical for understanding how mRNAs are regulated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). Here, we describe a method, poly(A) binding protein-mediated mRNA 3' end retrieval by crosslinking immunoprecipitation (PAPERCLIP), that shows high specificity for mRNA 3' ends and compares favorably with existing 3' end mapping methods. PAPERCLIP uncovers a previously unrecognized role of CstF64/64tau in promoting the usage of a selected group of non-canonical poly(A) sites, the majority of which contain a downstream GUKKU motif. Furthermore, in the mouse brain, PAPERCLIP discovers extended 3' UTR sequences harboring functional miRNA binding sites and reveals developmentally regulated APA shifts, including one in Atp2b2 that is evolutionarily conserved in humans and results in the gain of a functional binding site of miR-137. PAPERCLIP provides a powerful tool to decipher post-transcriptional regulation of mRNAs through APA in vivo.


Assuntos
Imunoprecipitação/métodos , MicroRNAs/metabolismo , Poli A/metabolismo , Proteínas tau/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Células Cultivadas , Sequência Conservada , Evolução Molecular , Células HEK293 , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos/genética , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
20.
Elife ; 52016 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-27223325

RESUMO

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.


Assuntos
Antígenos de Neoplasias/metabolismo , Orientação de Axônios , Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Camundongos , Camundongos Knockout , Antígeno Neuro-Oncológico Ventral
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