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1.
Mol Cell ; 77(5): 999-1013.e6, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017896

RESUMO

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.


Assuntos
Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Spliceossomos/metabolismo , Animais , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Transdução de Sinais , Espermatogênese/genética , Spliceossomos/genética
2.
Mol Cell ; 56(2): 298-310, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25263594

RESUMO

BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.


Assuntos
Processamento Alternativo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Proteínas Correpressoras , Metilação de DNA/genética , Proteínas de Ligação a DNA , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Íntrons/genética , Lisina/genética , Lisina/metabolismo , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Processamento Pós-Transcricional do RNA/genética , RNA Interferente Pequeno , Ribonucleoproteína Nuclear Pequena U5/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Análise de Sequência de RNA , Spliceossomos/genética
3.
J Biol Chem ; 294(28): 10998-11010, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31160337

RESUMO

The Y-box binding protein 1 (YB-1) is a member of the cold shock domain (CSD) protein family and is recognized as an oncogenic factor in several solid tumors. By binding to RNA, YB-1 participates in several steps of posttranscriptional regulation of gene expression, including mRNA splicing, stability, and translation; microRNA processing; and stress granule assembly. However, the mechanisms in YB-1-mediated regulation of RNAs are unclear. Previously, we used both systematic evolution of ligands by exponential enrichment (SELEX) and individual-nucleotide resolution UV cross-linking and immunoprecipitation coupled RNA-Seq (iCLIP-Seq) analyses, which defined the RNA-binding consensus sequence of YB-1 as CA(U/C)C. We also reported that through binding to its core motif CAUC in primary transcripts, YB-1 regulates the alternative splicing of a CD44 variable exon and the biogenesis of miR-29b-2 during both Drosha and Dicer steps. To elucidate the molecular basis of the YB-1-RNA interactions, we report high-resolution crystal structures of the YB-1 CSD in complex with different RNA oligos at 1.7 Å resolution. The structure revealed that CSD interacts with RNA mainly through π-π stacking interactions assembled by four highly conserved aromatic residues. Interestingly, YB-1 CSD forms a homodimer in solution, and we observed that two residues, Tyr-99 and Asp-105, at the dimer interface are important for YB-1 CSD dimerization. Substituting these two residues with Ala reduced CSD's RNA-binding activity and abrogated the splicing activation of YB-1 targets. The YB-1 CSD-RNA structures presented here at atomic resolution provide mechanistic insights into gene expression regulated by CSD-containing proteins.


Assuntos
Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/ultraestrutura , Processamento Alternativo/fisiologia , Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Éxons/fisiologia , Humanos , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Proteína 1 de Ligação a Y-Box/genética
4.
EMBO Rep ; 19(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29735517

RESUMO

Alternative pre-mRNA splicing plays important roles in regulating self-renewal and differentiation of embryonic stem cells (ESCs). However, how specific alternative splicing programs are established in ESCs remains elusive. Here, we show that a subset of alternative splicing events in ESCs is dependent on miR-294 expression. Remarkably, roughly 60% of these splicing events are affected by the depletion of Muscleblind-Like Splicing Regulator 1 and 2 (Mbnl1/2). Distinct from canonical miRNA function, miR-294 represses Mbnl1/2 through both posttranscriptional and epigenetic mechanisms. Furthermore, we uncover non-canonical functions of MBNL proteins that bind and promote the expression of miR-294 targets, including Cdkn1a and Tgfbr2, thereby opposing the role of miR-294 in regulating cell proliferation, apoptosis, and epithelial-mesenchymal transition (EMT). Our study reveals extensive interactions between miRNAs and splicing factors, highlighting their roles in regulating cell type-specific alternative splicing and defining gene expression programs during development and cellular differentiation.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/fisiologia , MicroRNAs/fisiologia , Proteínas de Ligação a RNA/fisiologia , Processamento Alternativo , Animais , Apoptose/genética , Linhagem Celular , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , MicroRNAs/genética
5.
Mol Cell ; 45(4): 459-69, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22264826

RESUMO

Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its MED23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit, MED23, but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally, MED23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events, as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that MED23 can regulate hnRNP L occupancy at their coregulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.


Assuntos
Processamento Alternativo , Complexo Mediador/fisiologia , RNA Mensageiro/metabolismo , Animais , Células Cultivadas , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/fisiologia , Camundongos , Modelos Genéticos , Poliadenilação
6.
Nucleic Acids Res ; 43(17): 8516-28, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26240386

RESUMO

Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteína 1 de Ligação a Y-Box/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Genoma Humano , Genômica , Glioblastoma/enzimologia , Glioblastoma/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Análise de Sequência de RNA
7.
PLoS Genet ; 10(4): e1004289, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24722255

RESUMO

Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.


Assuntos
Processamento Alternativo/genética , Neoplasias Pulmonares/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Regulação para Baixo/genética , Genes Supressores de Tumor , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Receptores Notch/genética , Transdução de Sinais/genética
8.
Nucleic Acids Res ; 40(17): 8622-36, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22730292

RESUMO

The human Y box-binding protein-1 (YB-1) is a deoxyribonucleic acid (DNA)/ribonucleic acid (RNA)-binding protein with pleiotropic functions. Besides its roles in the regulation of transcription and translation, several recent studies indicate that YB-1 is a spliceosome-associated protein and is involved in alternative splicing, but the underlying mechanism has remained elusive. Here, we define both CAUC and CACC as high-affinity binding motifs for YB-1 by systematic evolution of ligands by exponential enrichment (SELEX) and demonstrate that these newly defined motifs function as splicing enhancers. Interestingly, on the endogenous CD44 gene, YB-1 appears to mediate a network interaction to activate exon v5 inclusion via multiple CAUC motifs in both the alternative exon and its upstream polypyrimidine tract. We provide evidence that YB-1 activates splicing by facilitating the recruitment of U2AF65 to weak polypyrimidine tracts through direct protein-protein interactions. Together, these findings suggest a vital role of YB-1 in activating a subset of weak 3' splice sites in mammalian cells.


Assuntos
Processamento Alternativo , Éxons , Íntrons , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Proteínas Nucleares/química , Motivos de Nucleotídeos , Domínios e Motivos de Interação entre Proteínas , RNA/química , RNA/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Técnica de Seleção de Aptâmeros , Fator de Processamento U2AF , Proteína 1 de Ligação a Y-Box/química
9.
Hum Mol Genet ; 19(7): 1153-64, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20053671

RESUMO

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader-Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.


Assuntos
Processamento Alternativo , Síndrome de Prader-Willi/genética , RNA Nucleolar Pequeno , Receptor 5-HT2C de Serotonina/genética , Animais , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Camundongos , Edição de RNA , Precursores de RNA
10.
J Cachexia Sarcopenia Muscle ; 13(1): 728-742, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34877814

RESUMO

BACKGROUND: Most of the microRNAs (MiRs) involved in myogenesis are transcriptional regulated. The role of MiR biogenesis in myogenesis has not been characterized yet. RNA-binding protein Musashi 2 (Msi2) is considered to be one of the major drivers for oncogenesis and stem cell proliferation. The functions of Msi2 in myogenesis have not been explored yet. We sought to investigate Msi2-regulated biogenesis of MiRs in myogenesis and muscle stem cell (MuSC) ageing. METHODS: We detected the expression of Msi2 in MuSCs and differentiated myotubes by quantitative reverse transcription PCR (RT-qPCR) and western blot. Msi2-binding partner human antigen R (HuR) was identified by immunoprecipitation followed by mass spectrometry analysis. The cooperative binding of Msi2 and HuR on MiR7a-1 was analysed by RNA immunoprecipitation and electrophoresis mobility shift assays. The inhibition of the processing of pri-MiR7a-1 mediated by Msi2 and HuR was shown by Msi2 and HuR knockdown. Immunofluorescent staining, RT-qPCR and immunoblotting were used to characterize the function of MiR7a-1 in myogenesis. Msi2 and HuR up-regulate cryptochrome circadian regulator 2 (Cry2) via MiR7a-1 was confirmed by the luciferase assay and western blot. The post-transcriptional regulatory cascade was further confirmed by RNAi and overexpressing of Msi2 and HuR in MuSCs, and the in vivo function was characterized by histopathological and molecular biological methods in Msi2 knockout mice. RESULTS: We identified a post-transcription regulatory cascade governed by a pair of RNA-binding proteins Msi2 and HuR. Msi2 is enriched in differentiated muscle cells and promotes MuSC differentiation despite its pro-proliferation functions in other cell types. Msi2 works synergistically with another RNA-binding protein HuR to repress the biogenesis of MiR7a-1 in an Msi2 dose-dependent manner to regulate the translation of the key component of the circadian core oscillator complex Cry2. Down-regulation of Cry2 (0.6-fold, vs. control, P < 0.05) mediated by MiR7a-1 represses MuSC differentiation. The disruption of this cascade leads to differentiation defects of MuSCs. In aged muscles, Msi2 (0.3-fold, vs. control, P < 0.01) expression declined, and the Cry2 protein level also decreases (0.5-fold, vs. control, P < 0.05), suggesting that the disruption of the Msi2-mediated post-transcriptional regulatory cascade could attribute to the declined ability of muscle regeneration in aged skeletal muscle. CONCLUSIONS: Our findings have identified a new post-transcriptional cascade regulating myogenesis. The cascade is disrupted in skeletal muscle ageing, which leads to declined muscle regeneration ability.


Assuntos
MicroRNAs , Desenvolvimento Muscular , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo
11.
J Clin Invest ; 132(8)2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35239512

RESUMO

Y-box-binding protein 1 (YB-1) is a multifunctional RNA binding protein involved in virtually every step of RNA metabolism. However, the functions and mechanisms of YB-1 in one of the most aggressive cancers, glioblastoma, are not well understood. In this study, we found that YB-1 protein was markedly overexpressed in glioblastoma and acted as a critical activator of both mTORC1 and mTORC2 signaling. Mechanistically, YB-1 bound the 5'UTR of CCT4 mRNA to promote the translation of CCT4, a component of the CCT chaperone complex, that in turn activated the mTOR signaling pathway by promoting mLST8 folding. In addition, YB-1 autoregulated its own translation by binding to its 5'UTR, leading to sustained activation of mTOR signaling. In patients with glioblastoma, high protein expression of YB-1 correlated with increased expression of CCT4 and mLST8 and activated mTOR signaling. Importantly, the administration of RNA decoys specifically targeting YB-1 in a mouse xenograft model resulted in slower tumor growth and better survival. Taken together, these findings uncover a disrupted proteostasis pathway involving a YB-1/CCT4/mLST8/mTOR axis in promoting glioblastoma growth, suggesting that YB-1 is a potential therapeutic target for the treatment of glioblastoma.


Assuntos
Glioblastoma , Proteína 1 de Ligação a Y-Box , Regiões 5' não Traduzidas , Animais , Linhagem Celular Tumoral , Chaperonina com TCP-1 , Glioblastoma/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Homólogo LST8 da Proteína Associada a mTOR/genética , Homólogo LST8 da Proteína Associada a mTOR/metabolismo
12.
Genome Biol ; 22(1): 77, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33685485

RESUMO

BACKGROUND: A-to-I RNA editing diversifies the transcriptome and has multiple downstream functional effects. Genetic variation contributes to RNA editing variability between individuals and has the potential to impact phenotypic variability. RESULTS: We analyze matched genetic and transcriptomic data in 49 tissues across 437 individuals to identify RNA editing events that are associated with genetic variation. Using an RNA editing quantitative trait loci (edQTL) mapping approach, we identify 3117 unique RNA editing events associated with a cis genetic polymorphism. Fourteen percent of these edQTL events are also associated with genetic variation in their gene expression. A subset of these events are associated with genome-wide association study signals of complex traits or diseases. We determine that tissue-specific levels of ADAR and ADARB1 are able to explain a subset of tissue-specific edQTL events. We find that certain microRNAs are able to differentiate between the edited and unedited isoforms of their targets. Furthermore, microRNAs can generate an expression quantitative trait loci (eQTL) signal from an edQTL locus by microRNA-mediated transcript degradation in an editing-specific manner. By integrative analyses of edQTL, eQTL, and microRNA expression profiles, we computationally discover and experimentally validate edQTL-microRNA pairs for which the microRNA may generate an eQTL signal from an edQTL locus in a tissue-specific manner. CONCLUSIONS: Our work suggests a mechanism in which RNA editing variability can influence the phenotypes of complex traits and diseases by altering the stability and steady-state level of critical RNA molecules.


Assuntos
Marcação de Genes , Variação Genética , MicroRNAs/genética , Edição de RNA , RNA-Seq/métodos , Transcriptoma , Alelos , Biologia Computacional/métodos , Marcação de Genes/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Desequilíbrio de Ligação , Herança Multifatorial , Especificidade de Órgãos/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Interferência de RNA , Estabilidade de RNA
13.
Nat Commun ; 12(1): 5767, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599184

RESUMO

Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in young females. RTT is mainly caused by mutations found in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Despite extensive studies, the molecular mechanism underlying RTT pathogenesis is still poorly understood. Here, we report MeCP2 as a key subunit of a higher-order multiunit protein complex Rbfox/LASR. Defective MeCP2 in RTT mouse models disrupts the assembly of the MeCP2/Rbfox/LASR complex, leading to reduced binding of Rbfox proteins to target pre-mRNAs and aberrant splicing of Nrxns and Nlgn1 critical for synaptic plasticity. We further show that MeCP2 disease mutants display defective condensate properties and fail to promote phase-separated condensates with Rbfox proteins in vitro and in cultured cells. These data link an impaired function of MeCP2 with disease mutation in splicing control to its defective properties in mediating the higher-order assembly of the MeCP2/Rbfox/LASR complex.


Assuntos
Proteína 2 de Ligação a Metil-CpG/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Processamento de RNA/metabolismo , Síndrome de Rett/genética , Processamento Alternativo/genética , Animais , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Éxons/genética , Feminino , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/química , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Domínios Proteicos , Subunidades Proteicas/metabolismo
14.
J Mol Cell Biol ; 13(5): 347-360, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-33196842

RESUMO

Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.


Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a Calmodulina/genética , Citoesqueleto/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Éxons/genética , Genes Supressores de Tumor/fisiologia , Células HEK293 , Humanos , Íntrons/genética , Neoplasias Pulmonares/patologia , RNA Mensageiro/genética , Regulação para Cima/genética
15.
Elife ; 102021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34184986

RESUMO

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.


Assuntos
Regiões 5' não Traduzidas/genética , Fatores de Crescimento de Fibroblastos/genética , Deficiência Intelectual/genética , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Adolescente , Animais , Criança , Pré-Escolar , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Aprendizagem , Masculino , Memória , Camundongos , Camundongos Endogâmicos C57BL
16.
RNA ; 14(2): 284-96, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18073345

RESUMO

Alternative mRNA splicing patterns are determined by the combinatorial control of regulator proteins and their target RNA sequences. We have recently characterized human hnRNP L as a global regulator of alternative splicing, binding to diverse C/A-rich elements. To systematically identify hnRNP L target genes on a genome-wide level, we have combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, we describe 11 target genes of hnRNP L that were validated by RT-PCR and that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved.


Assuntos
Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/metabolismo , Motivos de Aminoácidos , Éxons , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Humanos , Íntrons , Poliadenilação , Interferência de RNA
17.
RNA Biol ; 7(1): 56-64, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19946215

RESUMO

Heterogeneous nuclear ribonucleoprotein (hnRNP) L can regulate alternative mRNA splicing in diverse ways, binding to exonic or intronic sites and acting as either an activator or repressor. To investigate the mechanistic basis of hnRNP L-regulated alternative splicing, we focus here on two specific cases of hnRNP L-dependent splice site recognition. First, in the case of TJP1 our microarray data had suggested that exon 20 inclusion is regulated by hnRNP L as a repressor. Here we demonstrate by mutational analysis that exon skipping is mediated by a short silencer sequence consisting of three hnRNP L high-score binding motifs located upstream of the 3' splice site of the regulated exon. UV crosslinking and immunoprecipitation experiments showed that hnRNP L binding interferes with 3' splice site recognition by U2AF65. Second, SLC2A2 contains a CA-repeat sequence close to the 5' splice site of the regulated exon 4. Using psoralen crosslinking, we demonstrate that hnRNP L represses splicing by preventing 5' splice site recognition of the U1 snRNP. In sum, our data provide new insights into the mechanisms of how hnRNP L-bound to intronic sites-regulates exon recognition.


Assuntos
Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Mamíferos/genética , Sítios de Splice de RNA/genética , Animais , Sequência de Bases , Éxons/genética , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Células HeLa , Humanos , Íntrons/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Precursores de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Elementos Silenciadores Transcricionais/genética , Fator de Processamento U2AF , Proteína da Zônula de Oclusão-1
18.
Sci China C Life Sci ; 52(3): 253-60, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19294350

RESUMO

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing.


Assuntos
Processamento Alternativo , Precursores de RNA/metabolismo , Splicing de RNA , Animais , Éxons , Previsões , Regulação da Expressão Gênica , Humanos , Íntrons , Mamíferos/genética , Mamíferos/metabolismo , Modelos Genéticos , Precursores de RNA/genética , Transdução de Sinais
19.
J Mol Cell Biol ; 11(10): 930-939, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31152582

RESUMO

RNA-binding proteins (RBPs) are key regulators in post-transcriptional control of gene expression. Mutations that alter their activity or abundance have been implicated in numerous diseases such as neurodegenerative disorders and various types of cancer. This highlights the importance of RBP proteostasis and the necessity to tightly control the expression levels and activities of RBPs. In many cases, RBPs engage in an auto-regulatory feedback by directly binding to and influencing the fate of their own mRNAs, exerting control over their own expression. For this feedback control, RBPs employ a variety of mechanisms operating at all levels of post-transcriptional regulation of gene expression. Here we review RBP-mediated autogenous feedback regulation that either serves to maintain protein abundance within a physiological range (by negative feedback) or generates binary, genetic on/off switches important for e.g. cell fate decisions (by positive feedback).


Assuntos
Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Humanos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
20.
FASEB J ; 21(7): 1556-64, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17264164

RESUMO

NO, the product of endothelial NOS (eNOS), is a major regulator of vascular homeostasis and a critical factor in preventing cardiovascular diseases. We previously established a positive correlation between the number of variable CA repeats in intron 13 of human eNOS and the risk of coronary artery disease, and demonstrated that these polymorphic CA repeats function as a length-dependent splicing enhancer. By 5'-RACE polymerase chain reaction (PCR), we detected three splice variants containing novel 3' splice sites within intron 13--termed eNOS13A, eNOS13B, and eNOS13C--which share the first 13 exons of human eNOS and the same polyadenylation site at the end of the novel exon. When translated, all these splice variants would result in truncated proteins lacking eNOS activity. Coexpression of full-length eNOS with eNOS13A diminished eNOS enzyme activity in COS-7 cells by formation of heterodimers. The splice variants were expressed in endothelial cells and various human tissues. Finally, we demonstrate, using minigene transfection, that the expression of the eNOS13A splice variant is increased with high CA repeat numbers in intron 13. These data suggest a new mechanism for the regulation of eNOS activity and NO production in the cardiovascular system by truncated, dominant-negative splice variants of human eNOS.


Assuntos
Processamento Alternativo , Íntrons , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Primers do DNA , Dimerização , Células HeLa , Humanos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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