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1.
Nat Commun ; 11(1): 4744, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958768

RESUMO

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.


Assuntos
Sítios de Splice de RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Cristalografia por Raios X , Éxons/genética , Humanos , Mutação , Neoplasias/química , Neoplasias/genética , Nucleotídeos , Motivo de Reconhecimento de RNA , Processamento de RNA/genética , Fator de Processamento U2AF/química , Dedos de Zinco
2.
Tokai J Exp Clin Med ; 45(3): 113-116, 2020 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-32901897

RESUMO

Mutations in the gene encoding epidermal growth factor receptor (EGFR) are the most frequent driver mutations in lung adenocarcinoma in Japan. Exon 19 deletion and L858R mutation in exon 21 are the most common EGFR mutations. Uncommon mutations, such as G719X, S768I, and L861Q, and compound mutations, combinations of 2 common or uncommon mutations, have also been reported. EGFR tyrosine kinase inhibitors (TKIs) are effective against cancers harboring common mutations; however, their efficacy against cancers with uncommon or compound mutations remains unclear. We report the case of a 67-year-old man with lung adenocarcinoma (clinical stage IIIA [cT1N2M0]), harboring an uncommon compound mutation, G719X and S768I. The cancer progressed within 2 months of initial chemoradiotherapy. Treatment with afatinib (40 mg/day) produced a partial response, which was maintained for 17 months with continued treatment. A literature review revealed that lung cancer with G719X/S768I compound mutation exhibited good response to EGFR-TKIs, even better than that of lung cancers with single uncommon mutations.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Afatinib/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Idoso , Receptores ErbB/genética , Éxons/genética , Deleção de Genes , Humanos , Masculino , Resultado do Tratamento
3.
Am J Hum Genet ; 107(3): 487-498, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32800095

RESUMO

The aggregation and joint analysis of large numbers of exome sequences has recently made it possible to derive estimates of intolerance to loss-of-function (LoF) variation for human genes. Here, we demonstrate strong and widespread coupling between genic LoF intolerance and promoter CpG density across the human genome. Genes downstream of the most CpG-rich promoters (top 10% CpG density) have a 67.2% probability of being highly LoF intolerant, using the LOEUF metric from gnomAD. This is in contrast to 7.4% of genes downstream of the most CpG-poor (bottom 10% CpG density) promoters. Combining promoter CpG density with exonic and promoter conservation explains 33.4% of the variation in LOEUF, and the contribution of CpG density exceeds the individual contributions of exonic and promoter conservation. We leverage this to train a simple and easily interpretable predictive model that outperforms other existing predictors and allows us to classify 1,760 genes-which are currently unascertained in gnomAD-as highly LoF intolerant or not. These predictions have the potential to aid in the interpretation of novel variants in the clinical setting. Moreover, our results reveal that high CpG density is not merely a generic feature of human promoters but is preferentially encountered at the promoters of the most selectively constrained genes, calling into question the prevailing view that CpG islands are not subject to selection.


Assuntos
Ilhas de CpG/genética , Genoma Humano/genética , Mutação com Perda de Função/genética , Regiões Promotoras Genéticas/genética , Metilação de DNA/genética , Éxons/genética , Humanos , RNA Polimerase II/genética , Sítio de Iniciação de Transcrição
4.
PLoS One ; 15(7): e0233583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735619

RESUMO

Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.


Assuntos
Éxons/genética , Proteína Huntingtina/genética , Elongação Traducional da Cadeia Peptídica , Agregados Proteicos/genética , RNA Mensageiro/genética , Ribossomos/metabolismo , Sequência de Bases , Células Epiteliais , Genes Reporter , Células HEK293 , Humanos , Proteína Huntingtina/biossíntese , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Repetições Minissatélites , Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
PLoS One ; 15(7): e0233582, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735620

RESUMO

The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, branch point to 3' splice site (BPS-3'SS) distance and splice site strengths, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.


Assuntos
Processamento Alternativo , Atresia das Cóanas/patologia , Surdez/congênito , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Ribonucleoproteína Nuclear Pequena U5/deficiência , Spliceossomos/fisiologia , Apoptose , Diferenciação Celular , Técnicas de Reprogramação Celular , Atresia das Cóanas/genética , Células Clonais , Surdez/genética , Surdez/patologia , Transição Epitelial-Mesenquimal , Éxons/genética , Face/embriologia , Facies , Feminino , Cabeça/embriologia , Cardiopatias Congênitas/genética , Humanos , Crista Neural/citologia , Regiões Promotoras Genéticas/genética , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Deleção de Sequência , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Via de Sinalização Wnt
6.
Cytogenet Genome Res ; 160(5): 238-244, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32659759

RESUMO

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome.


Assuntos
Processamento Alternativo/genética , Grupo com Ancestrais do Continente Asiático/genética , Colágeno Tipo IV/genética , Mutação , Nefrite Hereditária/genética , Sítios de Splice de RNA/genética , Adulto , Linhagem Celular , Criança , Pré-Escolar , Simulação por Computador , Éxons/genética , Feminino , Hematúria/genética , Humanos , Masculino , Linhagem , Proteinúria/genética
7.
BMC Evol Biol ; 20(1): 85, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664916

RESUMO

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in the active transportation of various endogenous or exogenous substances. Two ABCG2 gene subfamily members have been identified in birds. A detailed comparative study of the ABCG2 and ABCG2-like genes aid our understanding of their evolutionary history at the molecular level and provide a theoretical reference for studying the specific functions of ABCG2 and ABCG2-like genes in birds. RESULTS: We first identified 77 ABCG2/ABCG2-like gene sequences in the genomes of 41 birds. Further analysis showed that both the nucleic acid and amino acid sequences of ABCG2 and ABCG2-like genes were highly conserved and exhibited high homology in birds. However, significant differences in the N-terminal structure were found between the ABCG2 and ABCG2-like amino acid sequences. A selective pressure analysis showed that the ABCG2 and ABCG2-like genes were affected by purifying selection during the process of bird evolution. CONCLUSIONS: We believe that multiple members of the ABCG2 gene subfamily exist on chromosome 4 in the ancestors of birds. Over the long course of evolution, only the ABCG2 gene was retained on chromosome 4 in birds. The ABCG2-like gene on chromosome 6 might have originated from chromosome replication or fusion. The structural differences between the N terminus of ABCG2 protein and those of ABCG2-like proteins might lead to functional differences between the corresponding genes.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Aves/genética , Evolução Molecular , Homologia de Sequência de Aminoácidos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Cromossomos/genética , Sequência Conservada/genética , Éxons/genética , Regulação da Expressão Gênica , Genoma , Íntrons/genética , Família Multigênica , Fases de Leitura Aberta/genética , Fosforilação , Filogenia , Domínios Proteicos , Sítios de Splice de RNA/genética , Seleção Genética , Sintenia/genética
8.
Nat Commun ; 11(1): 3304, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620809

RESUMO

A main assumption of molecular population genetics is that genomic mutation rate does not depend on sequence function. Challenging this assumption, a recent study has found a reduction in the mutation rate in exons compared to introns in somatic cells, ascribed to an enhanced exonic mismatch repair system activity. If this reduction happens also in the germline, it can compromise studies of population genomics, including the detection of selection when using introns as proxies for neutrality. Here we compile and analyze published germline de novo mutation data to test if the exonic mutation rate is also reduced in germ cells. After controlling for sampling bias in datasets with diseased probands and extended nucleotide context dependency, we find no reduction in the mutation rate in exons compared to introns in the germline. Therefore, there is no evidence that enhanced exonic mismatch repair activity determines the mutation rate in germline cells.


Assuntos
Éxons/genética , Mutação em Linhagem Germinativa , Íntrons/genética , Taxa de Mutação , Algoritmos , Reparo de Erro de Pareamento de DNA/genética , Evolução Molecular , Células Germinativas/metabolismo , Humanos , Modelos Genéticos , Mutação , Sequenciamento Completo do Exoma/métodos
9.
Medicine (Baltimore) ; 99(28): e21160, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32664151

RESUMO

BACKGROUND: Previous studies have investigated the correlation between xeroderma pigmentosumcomplementation group C (XPC) variants and prostate adenocarcinoma (PA) risk. Nevertheless, research findings remain inconclusive. METHODS: We conducted a pooled analysis to obtain a more accurate estimation of the relationship on XPC exon15 Lys939Gln polymorphism with susceptibility to PA. Moreover, in silico tools were employed to investigate the effect of XPC expression on PA patients' survival time. RESULTS: A total of 4306 patients and 4779 control subjects were assessed. The overall results indicated that XPC Lys939Gln variant was associated with PA risk (recessive genetic model: odds ratio = 1.15, 95% confidence interval = 1.02-1.30, Pheterogeneity= .044, P = .021, I= 45.2), especially in Asian descendants. Population-based studies revealed similar results (odds ratio = 1.15, 95% confidence interval = 1.01-1.32, Pheterogeneity= .146, P = .040, I = 39.0). In silico tools showed that XPC expression in Caucasian patients was lower than in the normal group. No positive association was observed in African patients. PA subjects with high XPC expression had a longer overall survival time than low expression group. CONCLUSION: Our findings indicated that XPC Lys939Gln variant might contribute to increased PA susceptibility, especially for Asian patients.


Assuntos
Adenocarcinoma/genética , Proteínas de Ligação a DNA/genética , Éxons/genética , Predisposição Genética para Doença/genética , Neoplasias da Próstata/genética , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Grupo com Ancestrais do Continente Europeu/genética , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único , Fatores de Risco
10.
BMC Evol Biol ; 20(1): 66, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503430

RESUMO

BACKGROUND: Alternative splicing (AS) generates various transcripts from a single gene and thus plays a significant role in transcriptomic diversity and proteomic complexity. Alu elements are primate-specific transposable elements (TEs) and can provide a donor or acceptor site for AS. In a study on TE-mediated AS, we recently identified a novel AluSz6-exonized ACTR8 transcript of the crab-eating monkey (Macaca fascicularis). In the present study, we sought to determine the molecular mechanism of AluSz6 exonization of the ACTR8 gene and investigate its evolutionary and functional consequences in the crab-eating monkey. RESULTS: We performed RT-PCR and genomic PCR to analyze AluSz6 exonization in the ACTR8 gene and the expression of the AluSz6-exonized transcript in nine primate samples, including prosimians, New world monkeys, Old world monkeys, and hominoids. AluSz6 integration was estimated to have occurred before the divergence of simians and prosimians. The Alu-exonized transcript obtained by AS was lineage-specific and expressed only in Old world monkeys and apes, and humans. This lineage-specific expression was caused by a single G duplication in AluSz6, which provides a new canonical 5' splicing site. We further identified other alternative transcripts that were unaffected by the AluSz6 insertion. Finally, we observed that the alternative transcripts were transcribed into new isoforms with C-terminus deletion, and in silico analysis showed that these isoforms do not have a destructive function. CONCLUSIONS: The single G duplication in the TE sequence is the source of TE exonization and AS, and this mutation may suffer a different fate of ACTR8 gene expression during primate evolution.


Assuntos
Regulação da Expressão Gênica , Proteínas dos Microfilamentos/genética , Mutação , Primatas/genética , Processamento Alternativo , Elementos Alu/genética , Animais , Elementos de DNA Transponíveis/genética , Evolução Molecular , Éxons/genética , Humanos
11.
PLoS Genet ; 16(6): e1008864, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32584820

RESUMO

Cytosine methylation is an ancient epigenetic modification yet its function and extent within genomes is highly variable across eukaryotes. In mammals, methylation controls transposable elements and regulates the promoters of genes. In insects, DNA methylation is generally restricted to a small subset of transcribed genes, with both intergenic regions and transposable elements (TEs) depleted of methylation. The evolutionary origin and the function of these methylation patterns are poorly understood. Here we characterise the evolution of DNA methylation across the arthropod phylum. While the common ancestor of the arthropods had low levels of TE methylation and did not methylate promoters, both of these functions have evolved independently in centipedes and mealybugs. In contrast, methylation of the exons of a subset of transcribed genes is ancestral and widely conserved across the phylum, but has been lost in specific lineages. A similar set of genes is methylated in all species that retained exon-enriched methylation. We show that these genes have characteristic patterns of expression correlating to broad transcription initiation sites and well-positioned nucleosomes, providing new insights into potential mechanisms driving methylation patterns over hundreds of millions of years.


Assuntos
Artrópodes/genética , Metilação de DNA , Epigênese Genética , Evolução Molecular , Animais , Ilhas de CpG/genética , Elementos de DNA Transponíveis/genética , Éxons/genética , Filogenia , Regiões Promotoras Genéticas/genética
12.
PLoS Genet ; 16(6): e1008830, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502192

RESUMO

Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Códon de Terminação , Conjuntos de Dados como Assunto , Embrião não Mamífero , Éxons/genética , Redes Reguladoras de Genes/genética , Homozigoto , Humanos , Íntrons/genética , Camundongos , Músculo Esquelético/inervação , Mutagênese , Mutação , Crescimento Neuronal/genética , Proteínas Nucleares/genética , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Alinhamento de Sequência , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
13.
Am J Hum Genet ; 107(2): 196-210, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589925

RESUMO

A major question in human genetics is how sequence variants of broadly expressed genes produce tissue- and cell type-specific molecular phenotypes. Genetic variation of alternative splicing is a prevalent source of transcriptomic and proteomic diversity in human populations. We investigated splicing quantitative trait loci (sQTLs) in 1,209 samples from 13 human brain regions, using RNA sequencing (RNA-seq) and genotype data from the Genotype-Tissue Expression (GTEx) project. Hundreds of sQTLs were identified in each brain region. Some sQTLs were shared across brain regions, whereas others displayed regional specificity. These "regionally ubiquitous" and "regionally specific" sQTLs showed distinct positional distributions of single-nucleotide polymorphisms (SNPs) within and outside essential splice sites, respectively, suggesting their regulation by distinct molecular mechanisms. Integrating the binding motifs and expression patterns of RNA binding proteins with exon splicing profiles, we uncovered likely causal variants underlying brain region-specific sQTLs. Notably, SNP rs17651213 created a putative binding site for the splicing factor RBFOX2 and was associated with increased splicing of MAPT exon 3 in cerebellar tissues, where RBFOX2 was highly expressed. Overall, our study reveals a more comprehensive spectrum and regional variation of sQTLs in human brain and demonstrates that such regional variation can be used to fine map potential causal variants of sQTLs and their associated neurological diseases.


Assuntos
Encéfalo/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Processamento de RNA/genética , Éxons/genética , Humanos , Proteômica/métodos , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
14.
Nat Commun ; 11(1): 2845, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504065

RESUMO

Exonic splicing enhancers (ESEs) are enriched in exons relative to introns and bind splicing activators. This study considers a fundamental question of co-evolution: How did ESE motifs become enriched in exons prior to the evolution of ESE recognition? We hypothesize that the high exon to intron motif ratios necessary for ESE function were created by mutational bias coupled with purifying selection on the protein code. These two forces retain certain coding motifs in exons while passively depleting them from introns. Through the use of simulations, genomic analyses, and high throughput splicing assays, we confirm the key predictions of this hypothesis, including an overlap between protein and splicing information in ESEs. We discuss the implications of mutational bias as an evolutionary driver in other cis-regulatory systems.


Assuntos
Elementos Facilitadores Genéticos , Evolução Molecular , Éxons/genética , Genoma Humano , Processamento de RNA , Simulação por Computador , Genômica , Ensaios de Triagem em Larga Escala , Humanos , Íntrons/genética , Modelos Genéticos , Mutação
15.
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
16.
J Cancer Res Clin Oncol ; 146(9): 2329-2338, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32596787

RESUMO

With the development of antitumor therapies, different treatment methods including monotherapy and combined therapy have achieved clinical efficacy in advanced epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients. Exon 19 deletion (ex19del) and exon 21 L858R mutation are common sensitive subtypes of EGFR mutation. However, potential distinct mechanisms are found from several dimensions including molecular structures, biological behaviors, concomitant mutations, resistance mechanisms and tumor mutation burdens. More evidence indicates the prognostic difference of EGFR subgroups. This review focused on the progress of potential distinct mechanisms and outcomes in clinical trials of advanced NSCLC patients with ex19del or exon 21 L858R mutation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Éxons/genética , Neoplasias Pulmonares/genética , Mutação/genética , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Prognóstico
17.
J Vis Exp ; (159)2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32449741

RESUMO

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.


Assuntos
Células Cultivadas/química , Éxons/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Urina/química , Humanos , Transfecção
18.
Hum Genet ; 139(11): 1363-1379, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32424618

RESUMO

We report truncating de novo variants in specific exons of FBRSL1 in three unrelated children with an overlapping syndromic phenotype with respiratory insufficiency, postnatal growth restriction, microcephaly, global developmental delay and other malformations. The function of FBRSL1 is largely unknown. Interestingly, mutations in the FBRSL1 paralogue AUTS2 lead to an intellectual disability syndrome (AUTS2 syndrome). We determined human FBRSL1 transcripts and describe protein-coding forms by Western blot analysis as well as the cellular localization by immunocytochemistry stainings. All detected mutations affect the two short N-terminal isoforms, which show a ubiquitous expression in fetal tissues. Next, we performed a Fbrsl1 knockdown in Xenopus laevis embryos to explore the role of Fbrsl1 during development and detected craniofacial abnormalities and a disturbance in neurite outgrowth. The aberrant phenotype in Xenopus laevis embryos could be rescued with a human N-terminal isoform, while the long isoform and the N-terminal isoform containing the mutation p.Gln163* isolated from a patient could not rescue the craniofacial defects caused by Fbrsl1 depletion. Based on these data, we propose that the disruption of the validated N-terminal isoforms of FBRSL1 at critical timepoints during embryogenesis leads to a hitherto undescribed complex neurodevelopmental syndrome.


Assuntos
Deficiência Intelectual/genética , Linfocinas/genética , Mutação/genética , Anormalidades Múltiplas/genética , Adolescente , Animais , Criança , Éxons/genética , Humanos , Masculino , Fenótipo , Isoformas de Proteínas/genética , Síndrome , Fatores de Transcrição/genética
19.
RNA ; 26(9): 1216-1233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467309

RESUMO

In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.


Assuntos
Sítios de Ligação/genética , Ligação Proteica/genética , RNA/química , RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Linhagem Celular , Células Eucarióticas/metabolismo , Éxons/genética , Células HEK293 , Humanos , Imunoprecipitação/métodos , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética
20.
RNA ; 26(9): 1257-1267, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467311

RESUMO

During breast cancer metastasis, the developmental process epithelial-mesenchymal transition (EMT) is abnormally activated. Transcriptional regulatory networks controlling EMT are well-studied; however, alternative RNA splicing also plays a critical regulatory role during this process. A comprehensive understanding of alternative splicing (AS) and the RNA binding proteins (RBPs) that regulate it during EMT and their impact on breast cancer remains largely unknown. In this study, we annotated AS in the breast cancer TCGA data set and identified an AS signature that is capable of distinguishing epithelial and mesenchymal states of the tumors. This AS signature contains 25 AS events, among which nine showed increased exon inclusion and 16 showed exon skipping during EMT. This AS signature accurately assigns the EMT status of cells in the CCLE data set and robustly predicts patient survival. We further developed an effective computational method using bipartite networks to identify RBP-AS networks during EMT. This network analysis revealed the complexity of RBP regulation and nominated previously unknown RBPs that regulate EMT-associated AS events. This study highlights the importance of global AS regulation during EMT in cancer progression and paves the way for further investigation into RNA regulation in EMT and metastasis.


Assuntos
Processamento Alternativo/genética , Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal/genética , RNA/genética , Linhagem Celular Tumoral , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Humanos , Células MCF-7 , Proteínas de Ligação a RNA/genética
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