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1.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 2357-2360, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018480

RESUMO

In the past decades an extensive mathematical literature was developed to model and analyze gene networks under both deterministic and stochastic formalisms. However, such literature is predominantly focused to deal with the modeling of transcriptional and translational regulation, but results related to post-transcriptional regulation and its connection with transcriptional regulation are poorly investigated. However, it is becoming of paramount importance the need for modeling post-transcriptional regulation via splicing especially for minor organisms or viruses.The aim of this study is to propose a first general basic modeling scheme for modeling gene expression via alternative splicing and investigating the basic deterministic and stochastic features of the pre-mRNA, mRNAs and proteins under different biological conditions.This first study showed the dynamical properties of alternative splicing, the faster kinetics of the pre-mRNA compared to the mRNA and the importance to stochastically model gene networks when considering the post-transcriptional regulation.


Assuntos
Precursores de RNA , Processamento de RNA , Processamento Alternativo , Redes Reguladoras de Genes , Precursores de RNA/metabolismo , Processamento de RNA/genética , RNA Mensageiro/genética
2.
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
3.
Int J Oral Sci ; 12(1): 22, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737282

RESUMO

A splicing mutation in VPS4B can cause dentin dysplasia type I (DD-I), a hereditary autosomal-dominant disorder characterized by rootless teeth, the etiology of which is genetically heterogeneous. In our study, dental follicle cells (DFCs) were isolated and cultured from a patient with DD-I and compared with those from an age-matched, healthy control. In a previous study, this DD-I patient was confirmed to have a loss-of-function splicing mutation in VPS4B (IVS7 + 46C > G). The results from this study showed that the isolated DFCs were vimentin-positive and CK14-negative, indicating that the isolated cells were derived from the mesenchyme. DFCs harboring the VPS4B mutation had a significantly higher proliferation rate from day 3 to day 8 than control DFCs, indicating that VPS4B is involved in cell proliferation. The cells were then replenished with osteogenic medium to investigate how the VPS4B mutation affected osteogenic differentiation. Induction of osteogenesis, detected by alizarin red and alkaline phosphatase staining in vitro, was decreased in the DFCs from the DD-I patient compared to the control DFCs. Furthermore, we also found that the VPS4B mutation in the DD-I patient downregulated the expression of osteoblast-related genes, such as ALP, BSP, OCN, RUNX2, and their encoded proteins. These outcomes confirmed that the DD-I-associated VPS4B mutation could decrease the capacity of DFCs to differentiate during the mineralization process and may also impair physiological root formation and bone remodeling. This might provide valuable insights and implications for exploring the pathological mechanisms underlying DD-I root development.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Saco Dentário/citologia , Displasia da Dentina/genética , Displasia da Dentina/fisiopatologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Osteogênese/genética , Processamento de RNA/genética , Estudos de Casos e Controles , Diferenciação Celular/genética , Células Cultivadas , Displasia da Dentina/patologia , Humanos , Mutação/genética
4.
Nat Commun ; 11(1): 4140, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811829

RESUMO

Investigating the evolution of complex phenotypes and the underlying molecular bases of their variation is critical to understand how organisms adapt to their environment. Applying classical quantitative genetics on a segregating population derived from a Can-0xCol-0 cross, we identify the MADS-box transcription factor FLOWERING LOCUS M (FLM) as a player of the phenotypic variation in plant growth and color. We show that allelic variation at FLM modulates plant growth strategy along the leaf economics spectrum, a trade-off between resource acquisition and resource conservation, observable across thousands of plant species. Functional differences at FLM rely on a single intronic substitution, disturbing transcript splicing and leading to the accumulation of non-functional FLM transcripts. Associations between this substitution and phenotypic and climatic data across Arabidopsis natural populations, show how noncoding genetic variation at a single gene might be adaptive through pleiotropic effects.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Processamento de RNA/genética , Alelos , Arabidopsis/metabolismo , Evolução Molecular , Pleiotropia Genética , Variação Genética , Íntrons , Fenótipo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Locos de Características Quantitativas/genética , Temperatura
5.
Nat Commun ; 11(1): 3698, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703943

RESUMO

Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development.


Assuntos
Proteínas de Ligação a DNA/genética , Deficiência Intelectual/genética , Retardo Mental Ligado ao Cromossomo X/genética , Mutação/genética , Proteínas de Ligação a RNA/genética , Spliceossomos/metabolismo , Proteínas de Peixe-Zebra/genética , Adulto , Animais , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Família , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Células NIH 3T3 , Linhagem , Fenótipo , Transporte Proteico , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , Síndrome , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
PLoS Genet ; 16(7): e1008944, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730252

RESUMO

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.


Assuntos
Chlamydomonas reinhardtii/genética , Íntrons/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de RNA/genética , Regulação da Expressão Gênica de Plantas/genética , Microalgas/genética , Regiões Promotoras Genéticas , Transcriptoma/genética
7.
PLoS Biol ; 18(7): e3000782, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692742

RESUMO

Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/metabolismo , Desenvolvimento Vegetal/genética , Processamento de RNA/genética , Transcrição Genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Flores/fisiologia , Histonas/metabolismo , Mutação com Perda de Função , Lisina/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Fenótipo , Raízes de Plantas/citologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos
8.
Nucleic Acids Res ; 48(15): 8626-8644, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621609

RESUMO

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.


Assuntos
Proteínas de Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética , Sequência de Aminoácidos/genética , Núcleo Celular/genética , Éxons/genética , Técnicas de Inativação de Genes , Humanos , RNA Mensageiro/genética , Ribonucleoproteínas/genética
9.
Proc Natl Acad Sci U S A ; 117(27): 15799-15808, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571908

RESUMO

The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1 t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed autoregulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as noncanonical splicing regulators were greatly increased, most notably, rpl10a When the levels of NMD-susceptible rpl10a transcripts were artificially increased in zebrafish larvae, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extraribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.


Assuntos
Glutationa/análogos & derivados , Degradação do RNAm Mediada por Códon sem Sentido/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Linfócitos T/imunologia , Proteínas de Peixe-Zebra/genética , Animais , Códon sem Sentido/genética , Fertilização/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutationa/genética , Homozigoto , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , RNA Mensageiro/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Peixe-Zebra/genética
10.
Cell Prolif ; 53(8): e12861, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32596958

RESUMO

OBJECTIVES: Due to the limited evaluation of the prognostic value of RNA processing genes (RPGs), which are regulators of alternative splicing events (ASEs) that have been shown to be associated with tumour progression, this study sought to determine whether colorectal cancer (CRC) could be further stratified based on the expression pattern of RPGs. MATERIALS AND METHODS: The gene expression profiles of CRCs were collected from TCGA (training set) and three external validation cohorts, representing 1060 cases totally. Cox regression with least absolute shrinkage and selection operator (LASSO) penalty was used to develop an RNA processing gene index (RPGI) risk score. Kaplan-Meier curves, multivariate Cox regression and restricted mean survival (RMS) analyses were harnessed to evaluate the prognostic value of the RPGI. RESULTS: A 22-gene RPGI signature was developed, and its risk score served as a strong independent prognostic factor across all data sets when adjusted for major clinical variables. Moreover, ASEs for certain genes, such as FGFR1 and the RAS oncogene family, were significantly correlated with RPGI. Expression levels of genes involved in splicing- and tumour-associated pathways were significantly correlated with RPGI score. Furthermore, a combination of RPGI with age and tumour stage resulted in significantly improved prognostic accuracy. CONCLUSIONS: Our findings highlighted the prognostic value of RPGs for risk stratification of CRC patients and provide insights into specific ASEs associated with the development of CRC.


Assuntos
Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Transcriptoma/genética , Processamento Alternativo/genética , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica/métodos , Humanos , Prognóstico , Processamento de RNA/genética , RNA Mensageiro/genética
11.
Am J Hum Genet ; 107(1): 83-95, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32516569

RESUMO

Synonymous codon usage has been identified as a determinant of translational efficiency and mRNA stability in model organisms and human cell lines. However, whether natural selection shapes human codon content to optimize translation efficiency is unclear. Furthermore, aside from those that affect splicing, synonymous mutations are typically ignored as potential contributors to disease. Using genetic sequencing data from nearly 200,000 individuals, we uncover clear evidence that natural selection optimizes codon content in the human genome. In deriving intolerance metrics to quantify gene-level constraint on synonymous variation, we discover that dosage-sensitive genes, DNA-damage-response genes, and cell-cycle-regulated genes are particularly intolerant to synonymous variation. Notably, we illustrate that reductions in codon optimality in BRCA1 can attenuate its function. Our results reveal that synonymous mutations most likely play an underappreciated role in human variation.


Assuntos
Uso do Códon/genética , Genoma Humano/genética , Seleção Genética/genética , Códon/genética , Evolução Molecular , Humanos , Mutação/genética , Processamento de RNA/genética , Estabilidade de RNA/genética
12.
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
13.
RNA ; 26(10): 1400-1413, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32518066

RESUMO

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3' end and removal of the Lsm1 carboxy-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.


Assuntos
Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítios de Ligação/genética , Ligação Proteica/genética , RNA/genética , Proteínas de Ligação ao Cap de RNA/genética , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/genética , Spliceossomos/genética
14.
RNA ; 26(10): 1320-1333, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554554

RESUMO

Human CD4+ T cells are often subdivided into distinct subtypes, including Th1, Th2, Th17, and Treg cells, that are thought to carry out distinct functions in the body. Typically, these T-cell subpopulations are defined by the expression of distinct gene repertoires; however, there is variability between studies regarding the methods used for isolation and the markers used to define each T-cell subtype. Therefore, how reliably studies can be compared to one another remains an open question. Moreover, previous analysis of gene expression in CD4+ T-cell subsets has largely focused on gene expression rather than alternative splicing. Here we take a meta-analysis approach, comparing eleven independent RNA-seq studies of human Th1, Th2, Th17, and/or Treg cells to determine the consistency in gene expression and splicing within each subtype across studies. We find that known master-regulators are consistently enriched in the appropriate subtype; however, cytokines and other genes often used as markers are more variable. Importantly, we also identify previously unknown transcriptomic markers that appear to consistently differentiate between subsets, including a few Treg-specific splicing patterns. Together this work highlights the heterogeneity in gene expression between samples designated as the same subtype, but also suggests additional markers that can be used to define functional groupings.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Expressão Gênica/genética , Processamento de RNA/genética , Subpopulações de Linfócitos T/fisiologia , Transcriptoma/genética , Adulto , Células Cultivadas , Citocinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
15.
Nucleic Acids Res ; 48(13): 7066-7078, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32484558

RESUMO

During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.


Assuntos
Bases de Dados Genéticas , Evolução Molecular , Íntrons/genética , Processamento de RNA/genética , Spliceossomos/genética , Animais , Genoma , Humanos , Filogenia , Plantas/genética , RNA Longo não Codificante/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Leveduras/genética
16.
Nucleic Acids Res ; 48(12): 6889-6905, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479602

RESUMO

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.


Assuntos
Esclerose Amiotrófica Lateral/genética , Homeostase/genética , Proteína FUS de Ligação a RNA/genética , Animais , Citoplasma/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Íntrons/genética , Mutação com Perda de Função , Camundongos , Camundongos Knockout , Mutação/genética , Processamento de RNA/genética , Superóxido Dismutase-1/genética , Proteína com Valosina/genética
17.
Gene ; 754: 144861, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32531454

RESUMO

Alu sequences are the most abundant repetitive elements in the human genome, and have proliferated to more than one million copies in the human genome. Primate-specific Alu sequences account for ~10% of the human genome, and their spread within the genome has the potential to generate new exons. The new exons produced by Alu elements appear in various primate genes, and their functions have been elucidated. Here, we identified a new exon in the insulin-like 3 gene (INSL3), which evolved ~50 million years ago, and led to a splicing variant with 31 extra amino acid residues in addition to the original 95 nucleotides (NTs) of INSL3. The Alu-INSL3 isoform underwent diverse changes during primate evolution; we identified that human Alu-INSL3 might be on its way to functionality and has potential to antagonize LGR8-INSL3 function. Therefore, the present study is designed to provide an example of the evolutionary trajectory of a variant peptide hormone antagonist that caused by the insertion of an Alu element in primates.


Assuntos
Evolução Molecular , Insulina/genética , Primatas/genética , Proteínas/genética , Processamento de RNA/genética , Elementos Alu , Sequência de Aminoácidos , Animais , Sequência de Bases , Humanos , Isoformas de Proteínas , Homologia de Sequência
18.
Nucleic Acids Res ; 48(11): 6294-6309, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402057

RESUMO

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.


Assuntos
Pareamento de Bases , Éxons , Íntrons , Processamento de RNA , Adenoviridae/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Éxons/genética , Inativação Gênica , Íntrons/genética , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento de RNA/genética , Spliceossomos/metabolismo , Transcriptoma/genética , Globinas beta/genética
19.
Nucleic Acids Res ; 48(12): 6824-6838, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32432721

RESUMO

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.


Assuntos
Acetiltransferases/genética , Dengue/genética , Imunidade Inata/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Apoptose/genética , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Éxons/genética , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Isoformas de Proteínas/genética , Processamento de RNA/genética , RNA-Seq , Replicação Viral/genética
20.
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
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