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1.
Nat Commun ; 12(1): 1648, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712605

RESUMO

Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/genética , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma
2.
Nat Commun ; 12(1): 1488, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674615

RESUMO

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.


Assuntos
Éxons , RNA Helicases/química , RNA Helicases/metabolismo , Precursores de RNA/metabolismo , Processamento de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , Precursores de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Fúngico/metabolismo , Proteínas Recombinantes , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/química
3.
Life Sci ; 273: 119258, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33636176

RESUMO

BACKGROUND: As the most prevalent post-transcriptional mRNA modification in eukaryotes, N6-Methyladenosine (m6A) is closely linked to the occurrence and development of colorectal cancer (CRC). However, there is no systematic evaluation of the expression of m6A regulatory genes in CRC. METHODS: By analyzing the TCGA database, we identified METTL3, YTHDF1, IGF2BP1, IGF2BP3, EIF3B, HNRNPA2B1 as overexpressed m6A regulators in CRC. After verification by immunohistochemistry (IHC) in 10 CRC cases, YTHDF1, IGF2BP1, IGF2BP3, and EIF3B were identified as potential biomarkers in CRC. Further validation was done by IHC and qRT-PCR in two larger cohorts. RESULTS: We identified 6 up-regulated m6A regulatory genes in CRC in TCGA analysis, and verified that YTHDF1, IGF2BP1, IGF2BP3, and EIFB3 were all significantly differentially expressed between CRC and normal tissues by IHC (p < 0.0001). In another larger cohort, we further validated the overexpression of those genes in CRC as compared to both normal tissues (p < 0.0001) and adenoma tissues (p < 0.05). Detailed analysis suggested that detection of one of the three genes, YTHDF1, IGF2BP1 and IGF2BP3, and combined detection of EIF3B gene could be a good strategy for early diagnosis of both CRC and precancerous lesions. Furthermore, we found that the mRNA levels of YTHDF1, IGF2BP1, and IGF2BP3 were also significantly up-regulated in CRC but not adenoma as compared to normal tissues. CONCLUSION: We evaluted the abnormal expression of m6A regulatory genes during CRC carcinogenesis, and identified four m6A genes (YTHDF1, IGF2BP1, IGF2BP3, and EIF3B) as potential biomarkers of both CRC and adenoma.


Assuntos
Adenocarcinoma/patologia , Adenoma/patologia , Adenosina/análogos & derivados , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/química , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenosina/química , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Prognóstico , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
4.
Nucleic Acids Res ; 49(5): 2460-2487, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33550394

RESUMO

Ca2+-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH and ATP supply by mutually exclusive OGDH exons 4a and 4b. Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5' splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5' splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca2+ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca2+-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.


Assuntos
Processamento Alternativo , Regulação da Temperatura Corporal/genética , Íntrons , Complexo Cetoglutarato Desidrogenase/genética , Animais , Cálcio/metabolismo , Evolução Molecular , Éxons , Células HEK293 , Humanos , Sequências Repetitivas Dispersas , Complexo Cetoglutarato Desidrogenase/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de RNA/química , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Vertebrados/genética
5.
Nucleic Acids Res ; 49(5): 2509-2521, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33555349

RESUMO

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.


Assuntos
Chaperonas Moleculares/genética , Proteínas Mutantes Quiméricas/genética , Neuroblastoma/genética , Processamento de RNA , Spliceossomos/efeitos dos fármacos , Aminoaciltransferases/metabolismo , Animais , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Fusão Gênica , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Camundongos Nus , Chaperonas Moleculares/metabolismo , Proteínas Mutantes Quiméricas/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Deleção de Sequência , Fatores de Transcrição/metabolismo , Proteínas tau/metabolismo
6.
Nucleic Acids Res ; 49(3): 1688-1707, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33444449

RESUMO

Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mutação , Estabilidade Proteica , Processamento de RNA , Transcriptoma
7.
Science ; 371(6535)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33509932

RESUMO

The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP. Our study provides a framework for the mechanistic understanding of the function of the human minor spliceosome.


Assuntos
Spliceossomos/química , Spliceossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Microscopia Crioeletrônica , Ciclofilinas/química , Ciclofilinas/metabolismo , Humanos , Íntrons , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , Precursores de RNA/química , Precursores de RNA/metabolismo , Processamento de RNA , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo
8.
Nucleic Acids Res ; 49(2): 636-645, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33337476

RESUMO

Phase-separated membraneless bodies play important roles in nucleic acid biology. While current models for the roles of phase separation largely focus on the compartmentalization of constituent proteins, we reason that other properties of phase separation may play functional roles. Specifically, we propose that interfaces of phase-separated membraneless bodies could have functional roles in spatially organizing biochemical reactions. Here we propose such a model for the nuclear speckle, a membraneless body implicated in RNA splicing. In our model, sequence-dependent RNA positioning along the nuclear speckle interface coordinates RNA splicing. Our model asserts that exons are preferentially sequestered into nuclear speckles through binding by SR proteins, while introns are excluded through binding by nucleoplasmic hnRNP proteins. As a result, splice sites at exon-intron boundaries are preferentially positioned at nuclear speckle interfaces. This positioning exposes splice sites to interface-localized spliceosomes, enabling the subsequent splicing reaction. Our model provides a simple mechanism that seamlessly explains much of the complex logic of splicing. This logic includes experimental results such as the antagonistic duality between splicing factors, the position dependence of splicing sequence motifs, and the collective contribution of many motifs to splicing decisions. Similar functional roles for phase-separated interfaces may exist for other membraneless bodies.


Assuntos
Núcleo Celular/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Modelos Genéticos , Processamento de RNA , Sequência de Bases , Núcleo Celular/metabolismo , Éxons , Humanos , Motivos de Nucleotídeos , Ligação Proteica , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo
9.
Cancer Lett ; 496: 72-83, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33038489

RESUMO

Splicing alterations represent an actionable cancer hallmark. Splicing factor 3B subunit 1 (SF3B1) is a crucial splicing factor that can be targeted pharmacologically (e.g. pladienolide-B). Here, we show that SF3B1 is overexpressed (RNA/protein) in hepatocellular carcinoma (HCC) in two retrospective (n = 154 and n = 172 samples) and in five in silico cohorts (n > 900 samples, including TCGA) and that its expression is associated with tumor aggressiveness, oncogenic splicing variants expression (KLF6-SV1, BCL-XL) and decreased overall survival. In vitro, SF3B1 silencing reduced cell viability, proliferation and migration and its pharmacological blockade with pladienolide-B inhibited proliferation, migration, and formation of tumorspheres and colonies in liver cancer cell lines (HepG2, Hep3B, SNU-387), whereas its effects on normal-like hepatocyte-derived THLE-2 proliferation were negligible. Pladienolide-B also reduced the in vivo growth and the expression of tumor-markers in Hep3B-induced xenograft tumors. Moreover, SF3B1 silencing and/or blockade markedly modulated the activation of key signaling pathways (PDK1, GSK3b, ERK, JNK, AMPK) and the expression of cancer-associated genes (CDK4, CD24) and oncogenic SVs (KLF6-SV1). Therefore, the genetic and/or pharmacological inhibition of SF3B1 may represent a promising novel therapeutic strategy worth to be explored through randomized controlled trials.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Adulto , Idoso , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosfoproteínas/genética , Prognóstico , Fatores de Processamento de RNA/genética , Estudos Retrospectivos , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Biomed Pharmacother ; 133: 111075, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378974

RESUMO

N6-methyladenosine (m6A) is one of the most abundant messenger RNAs modification. Increasing evidence illustrates its critical role on gastric cancer. Here, present research focuses on the potential function of m6A methyltransferase Wilms' tumour 1-associated protein (WTAP) in gastric cancer tumorigenesis. Firstly, m6A immunoprecipitation sequencing analysis (MeRIP-Seq) analysis demonstrated the m6A profile in gastric cancer cells. Both WTAP and the m6A expression were up-regulated in gastric cancer tissue and cells. The high-expression of WTAP was closely correlated with poor prognosis of gastric cancer patients. Functional experiments illustrated that WTAP promoted the proliferation and glycolytic capacity (glucose uptake, lactate production and extracellular acidification rate) in vitro, and the knockdown of WTAP suppressed the tumor growth in vivo. Mechanistically, HK2 was identified to be the target of WTAP using MeRIP-Seq and MeRIP-qPCR. WTAP enhanced the stability of HK2 mRNA through binding with the 3'-UTR m6A site. In conclusion, our results demonstrate the oncogenic role of WTAP and its m6A-mediated regulation on gastric cancer Warburg effect, providing a novel approach and therapeutic target in gastric cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Hexoquinase/metabolismo , Fatores de Processamento de RNA/metabolismo , Neoplasias Gástricas/enzimologia , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Estabilidade Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Hexoquinase/genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fatores de Processamento de RNA/genética , Estabilidade de RNA , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Carga Tumoral
11.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348896

RESUMO

SF3B1 is a core component of the U2 spliceosome that is frequently mutated in cancer. We have previously shown that titrating the activity of SF3B1, using the inhibitor pladienolide B (PB), affects distinct steps of the heat shock response (HSR). Here, we identify other genes that are sensitive to different levels of SF3B1 (5 vs. 100 nM PB) using RNA sequencing. Significant changes to mRNA splicing were identified at both low PB and high PB concentrations. Changes in expression were also identified in the absence of alternative splicing, suggesting that SF3B1 influences other gene expression pathways. Surprisingly, gene expression changes identified in low PB are not predictive of changes in high PB. Specific pathways were identified with differential sensitivity to PB concentration, including nonsense-mediated decay and protein-folding homeostasis, both of which were validated using independent reporter constructs. Strikingly, cells exposed to low PB displayed enhanced protein-folding capacity relative to untreated cells. These data reveal that the transcriptome is exquisitely sensitive to SF3B1 and suggests that the activity of SF3B1 is finely regulated to coordinate mRNA splicing, gene expression and cellular physiology.


Assuntos
Processamento Alternativo , Fenômenos Fisiológicos Celulares , Regulação da Expressão Gênica/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Células HEK293 , Humanos , Macrolídeos/farmacologia , Degradação do RNAm Mediada por Códon sem Sentido , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , RNA Mensageiro/genética
12.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333932

RESUMO

Uveal melanoma (UM) is the most common primary intraocular malignancy of the eye. It has a high metastatic potential and mainly spreads to the liver. Genetics play a vital role in tumor classification and prognostication of UM metastatic disease. One of the driver genes mutated in metastasized UM is subunit 1 of splicing factor 3b (SF3B1), a component of the spliceosome complex. Recurrent mutations in components of the spliceosome complex are observed in UM and other malignancies, suggesting an important role in tumorigenesis. SF3B1 is the most common mutated spliceosome gene and in UM it is associated with late-onset metastasis. This review summarizes the genetic and epigenetic insights of spliceosome mutations in UM. They form a distinct subgroup of UM and have similarities with other spliceosome mutated malignancies.


Assuntos
Melanoma/genética , Mutação , Fatores de Processamento de RNA/genética , Neoplasias Uveais/genética , Substituição de Aminoácidos , Éxons , Frequência do Gene , Humanos , Melanoma/metabolismo , Melanoma/mortalidade , Melanoma/patologia , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento de RNA , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos , Telômero/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/mortalidade , Neoplasias Uveais/patologia
13.
Mol Cell ; 80(1): 127-139.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007253

RESUMO

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.


Assuntos
Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Animais , Catálise , Células HeLa , Humanos , Íntrons/genética , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estabilidade Proteica , RNA/química , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fatores de Tempo
14.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Am J Psychiatry ; 177(9): 855-866, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32600152

RESUMO

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a highly heritable psychiatric disorder. The objective of this study was to define ADHD-associated candidate genes and their associated molecular modules and biological themes, based on the analysis of rare genetic variants. METHODS: The authors combined data from 11 published copy number variation studies in 6,176 individuals with ADHD and 25,026 control subjects and prioritized genes by applying an integrative strategy based on criteria including recurrence in individuals with ADHD, absence in control subjects, complete coverage in copy number gains, and presence in the minimal region common to overlapping copy number variants (CNVs), as well as on protein-protein interactions and information from cross-species genotype-phenotype annotation. RESULTS: The authors localized 2,241 eligible genes in the 1,532 reported CNVs, of which they classified 432 as high-priority ADHD candidate genes. The high-priority ADHD candidate genes were significantly coexpressed in the brain. A network of 66 genes was supported by ADHD-relevant phenotypes in the cross-species database. Four significantly interconnected protein modules were found among the high-priority ADHD genes. A total of 26 genes were observed across all applied bioinformatic methods. Lookup in the latest genome-wide association study for ADHD showed that among those 26 genes, POLR3C and RBFOX1 were also supported by common genetic variants. CONCLUSIONS: Integration of a stringent filtering procedure in CNV studies with suitable bioinformatics approaches can identify ADHD candidate genes at increased levels of credibility. The authors' analytic pipeline provides additional insight into the molecular mechanisms underlying ADHD and allows prioritization of genes for functional validation in validated model organisms.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , RNA Polimerase III , Fatores de Processamento de RNA , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Variações do Número de Cópias de DNA/fisiologia , Bases de Dados Genéticas , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Humanos , Mapeamento de Interação de Proteínas/métodos , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
16.
Nature ; 583(7815): 310-313, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32494006

RESUMO

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52-7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.


Assuntos
Microscopia Crioeletrônica , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/ultraestrutura , Sequência de Bases , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Transativadores/química , Transativadores/metabolismo
17.
Nat Commun ; 11(1): 2973, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532987

RESUMO

Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.


Assuntos
Processamento Alternativo , Sistemas CRISPR-Cas/genética , Éxons/genética , Fatores de Processamento de RNA/genética , RNA/genética , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , RNA/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
18.
Am J Otolaryngol ; 41(4): 102547, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474328

RESUMO

BACKGROUND: N6-Methyladenosine (m6A) is a ubiquitous RNA modification with vital roles in various cancers, but little is known about its role in papillary thyroid carcinoma (PTC), a common endocrine malignancy. METHODS: In this study, an m6A RNA methylation regulator-based biomarker signature was developed for the effective prediction of prognosis in patients with PTC. The gene expression profiles of m6A RNA methylation regulators and the corresponding clinical information was downloaded from The Cancer Genome Atlas (TCGA). Differentially expressed m6A RNA methylation regulators between tumor and normal control samples, and correlation expression levels, clinical parameters, and outcomes were evaluated. And a prognostic signature was built using a PTC cohort from TCGA. RESULTS: The expression level of HNRNPC was remarkably upregulated in tumor samples, while WTAP, RBM15, YTHDC2, YTHDC1, FTO, METTL14, METTL3, ALKBH5, KIAA1429, YTHDF1, and ZC3H13 were significantly downregulated in the cancer specimens compared with those in control samples. A three-gene prognostic signature comprising RBM15, KIAA1429, and FTO could predict overall survival in patients with PTC. In addition, the prognostic signature-based risk score was identified as an independent prognostic indicator for PTC. CONCLUSIONS: We established a robust m6A RNA methylation regulator-based molecular signature for predicting prognosis in patients with PTC with high accuracy; this signature might provide important guidance for therapeutic strategies.


Assuntos
Regulação Neoplásica da Expressão Gênica , Expressão Gênica , Metiltransferases/genética , Metiltransferases/metabolismo , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Idoso , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Prognóstico , RNA Helicases/genética , RNA Helicases/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Taxa de Sobrevida , Câncer Papilífero da Tireoide/mortalidade , Neoplasias da Glândula Tireoide/mortalidade , Regulação para Cima/genética
19.
Nat Commun ; 11(1): 2506, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427851

RESUMO

The genetic basis and corresponding clinical relevance of prolactinomas remain poorly understood. Here, we perform whole genome sequencing (WGS) on 21 patients with prolactinomas to detect somatic mutations and then validate the mutations with digital polymerase chain reaction (PCR) analysis of tissue samples from 227 prolactinomas. We identify the same hotspot somatic mutation in splicing factor 3 subunit B1 (SF3B1R625H) in 19.8% of prolactinomas. These patients with mutant prolactinomas display higher prolactin (PRL) levels (p = 0.02) and shorter progression-free survival (PFS) (p = 0.02) compared to patients without the mutation. Moreover, we identify that the SF3B1R625H mutation causes aberrant splicing of estrogen related receptor gamma (ESRRG), which results in stronger binding of pituitary-specific positive transcription factor 1 (Pit-1), leading to excessive PRL secretion. Thus our study validates an important mutation and elucidates a potential mechanism underlying the pathogenesis of prolactinomas that may lead to the development of targeted therapeutics.


Assuntos
Fosfoproteínas/genética , Prolactinoma/genética , Fatores de Processamento de RNA/genética , Adulto , Feminino , Humanos , Masculino , Mutação , Fosfoproteínas/metabolismo , Intervalo Livre de Progressão , Prolactina/genética , Prolactina/metabolismo , Prolactinoma/metabolismo , Prolactinoma/mortalidade , Fatores de Processamento de RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Fator de Transcrição Pit-1/genética , Fator de Transcrição Pit-1/metabolismo , Adulto Jovem
20.
Nucleic Acids Res ; 48(10): 5656-5669, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329777

RESUMO

Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here, we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell's transcriptional regime.


Assuntos
Acetilglucosamina/metabolismo , Glicosídeo Hidrolases/genética , Íntrons , N-Acetilglucosaminiltransferases/genética , Processamento de RNA , Linhagem Celular , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA-Seq
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