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1.
Nat Commun ; 12(1): 335, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436550

RESUMO

Previous transcriptomic profiling studies have typically focused on separately analyzing mRNA expression, alternative splicing and alternative polyadenylation differences between cell and tissue types. However, the relative contribution of these three transcriptomic regulatory layers to cell type specification is poorly understood. This question is particularly relevant to neurons, given their extensive heterogeneity associated with brain location, morphology and function. In the present study, we generated profiles for the three regulatory layers from developmentally and regionally distinct subpopulations of neurons from the mouse hippocampus and broader nervous system. Multi-omics factor analyses revealed differing contributions of each transcriptomic layer in the discrimination of neurons based on their stage of development, region, and function. Importantly, profiles of differential alternative splicing and polyadenylation better discriminated specific neuronal subtype populations than gene expression patterns. These results provide evidence for differential relative contributions of coordinated gene regulatory layers in the specification of neuronal subtypes.


Assuntos
Regulação da Expressão Gênica , Neurônios/metabolismo , Transcriptoma/genética , Regiões 3' não Traduzidas/genética , Processamento Alternativo/genética , Animais , Regulação para Baixo/genética , Hipocampo/anatomia & histologia , Hipocampo/citologia , Camundongos , Poliadenilação/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição Genética , Regulação para Cima/genética
2.
Nat Commun ; 12(1): 266, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436621

RESUMO

Circular RNAs (circRNAs) have emerged as an important class of functional RNA molecules. Short-read RNA sequencing (RNA-seq) is a widely used strategy to identify circRNAs. However, an inherent limitation of short-read RNA-seq is that it does not experimentally determine the full-length sequences and exact exonic compositions of circRNAs. Here, we report isoCirc, a strategy for sequencing full-length circRNA isoforms, using rolling circle amplification followed by nanopore long-read sequencing. We describe an integrated computational pipeline to reliably characterize full-length circRNA isoforms using isoCirc data. Using isoCirc, we generate a comprehensive catalog of 107,147 full-length circRNA isoforms across 12 human tissues and one human cell line (HEK293), including 40,628 isoforms ≥500 nt in length. We identify widespread alternative splicing events within the internal part of circRNAs, including 720 retained intron events corresponding to a class of exon-intron circRNAs (EIciRNAs). Collectively, isoCirc and the companion dataset provide a useful strategy and resource for studying circRNAs in human transcriptomes.


Assuntos
Bases de Dados Genéticas , Isoformas de RNA/genética , RNA Circular/genética , Transcriptoma/genética , Processamento Alternativo/genética , Células HEK293 , Humanos , RNA-Seq
3.
Nat Commun ; 12(1): 89, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397958

RESUMO

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.


Assuntos
Processamento Alternativo/genética , Desenvolvimento Muscular/genética , Contração Miocárdica/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Actinina/genética , Animais , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética
4.
Gene ; 766: 145146, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32941952

RESUMO

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many diseases, including Alzheimer's disease (AD). In the presented study, we have examined the influence of cellular hypoxia on mRNA splice variant formation from Alzheimer's disease-related Tau and APP genes in brain cells. We have shown that the hypoxic microenvironment influenced the formation of Tau mRNA splice variants, but had no effect on APP mRNA splice variant formation. Additionally, our presented results indicate that splicing factor SRSF1 but not SRSF5 alters the formation of Tau cellular mRNA splice variants in hypoxic cells. Obtained results have also shown that hypoxic brain cells possess enhanced CLK1-4 kinase mRNA levels. This study underlines that cellular hypoxia can influence disease development through changing pre-mRNA splicing.


Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Hipóxia Celular/genética , RNA Mensageiro/genética , Proteínas tau/genética , Processamento Alternativo/genética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Íntrons/genética , Precursores de RNA/genética , Transcrição Genética/genética
5.
Nucleic Acids Res ; 49(1): 479-490, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330934

RESUMO

The mammalian Ate1 gene encodes an arginyl transferase enzyme with tumor suppressor function that depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. We report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore RNA structure revert splicing to that of the wild type. In the endogenous Ate1 pre-mRNA, blocking the competing base pairings by LNA/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of R2R5 interaction changes the ratio of MXE. That is, Ate1 splicing is controlled by two independent, dynamically interacting, and functionally distinct RNA structure modules. Exon 7a becomes more included in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted, indicating that exon 7a/7b ratio depends on co-transcriptional RNA folding. In sum, these results demonstrate that splicing is coordinated both in time and in space over very long distances, and that the interaction of these components is mediated by RNA structure.


Assuntos
Processamento Alternativo/genética , Aminoaciltransferases/genética , Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Dobramento de RNA , Precursores de RNA/genética , RNA Mensageiro/genética , Células A549 , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Éxons/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Elongação da Transcrição Genética
6.
Mol Cell ; 80(1): 140-155.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007254

RESUMO

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Neurônios/metabolismo , Processamento Alternativo/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas ELAV/química , Larva/metabolismo , Mutação/genética , Poli A/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Nucleic Acids Res ; 48(18): 10241-10258, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941642

RESUMO

Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.


Assuntos
Chaperonas de Histonas/genética , Metiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética/genética , Fatores de Elongação da Transcrição/genética , Processamento Alternativo/genética , Regulação Fúngica da Expressão Gênica/genética , Código das Histonas/genética , Saccharomyces cerevisiae/genética
8.
DNA Cell Biol ; 39(11): 2040-2051, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32915082

RESUMO

As suggested by an increasing amount of evidence, there is alternative splicing (AS) modification within malignancy, which is related to cancer occurrence and development. AS within acute myeloid leukemia (AML) has not yet been systematically analyzed yet. This study analyzed the transcriptomic profiling and corresponding clinical data from AML cases based on The Cancer Genome Atlas (TCGA). In addition, the prediction model, along with the splicing network, was used to analyze the prognosis for AML patients according to the seven different AS event types. Among the 34,984 AS events across the 8830 genes, 2896 AS events were detected among 1905 genes, showing marked correlation with the overall survival of patients. The risk scoring model based on all AS event types was the most efficient in identifying the prognosis for AML patients. Meanwhile, the area under the curve at 1-, 3-, 5-year were 0.852, 0.935, 0.955, respectively. At the same time, the splicing regulating network, which was constituted by 21 splicing factor genes as well as 32 AS events related to survival, was characterized. In conclusion, our predictive model constructed based on the AS events accurately predicts the survival for AML patients. In addition, the network between AS events and splicing factor is established, which may serve as a potential mechanism.


Assuntos
Processamento Alternativo/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Análise de Sobrevida , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes , Humanos , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/patologia , Masculino , Prognóstico , RNA Mensageiro/genética , Transcriptoma/genética
9.
PLoS Comput Biol ; 16(9): e1008195, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898151

RESUMO

We present VALERIE (Visualising alternative splicing events from single-cell ribonucleic acid-sequencing experiments), an R package for visualising alternative splicing events at single-cell resolution. To explore any given specified genomic region, corresponding to an alternative splicing event, VALERIE generates an ensemble of informative plots to visualise cell-to-cell heterogeneity of alternative splicing profiles across single cells and performs statistical tests to compare percent spliced-in (PSI) values across the user-defined groups of cells. Among the features available, VALERIE displays PSI values, in lieu of read coverage, which is more suitable for representing alternative splicing profiles for a large number of samples typically generated by single-cell RNA-sequencing experiments. VALERIE is available on the Comprehensive R Archive Network (CRAN): https://cran.r-project.org/web/packages/VALERIE/index.html.


Assuntos
Processamento Alternativo/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Software , Animais , Células Cultivadas , Biologia Computacional , Camundongos
10.
Am J Hum Genet ; 107(3): 461-472, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32781045

RESUMO

RNA sequencing (RNA-seq) is a powerful technology for studying human transcriptome variation. We introduce PAIRADISE (Paired Replicate Analysis of Allelic Differential Splicing Events), a method for detecting allele-specific alternative splicing (ASAS) from RNA-seq data. Unlike conventional approaches that detect ASAS events one sample at a time, PAIRADISE aggregates ASAS signals across multiple individuals in a population. By treating the two alleles of an individual as paired, and multiple individuals sharing a heterozygous SNP as replicates, we formulate ASAS detection using PAIRADISE as a statistical problem for identifying differential alternative splicing from RNA-seq data with paired replicates. PAIRADISE outperforms alternative statistical models in simulation studies. Applying PAIRADISE to replicate RNA-seq data of a single individual and to population-scale RNA-seq data across many individuals, we detect ASAS events associated with genome-wide association study (GWAS) signals of complex traits or diseases. Additionally, PAIRADISE ASAS analysis detects the effects of rare variants on alternative splicing. PAIRADISE provides a useful computational tool for elucidating the genetic variation and phenotypic association of alternative splicing in populations.


Assuntos
Processamento Alternativo/genética , Predisposição Genética para Doença , Herança Multifatorial/genética , Transcriptoma/genética , Alelos , Feminino , Perfilação da Expressão Gênica , Genética Populacional/métodos , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Modelos Estatísticos , RNA-Seq , Sequenciamento Completo do Exoma
11.
Gene ; 760: 145021, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32763489

RESUMO

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.


Assuntos
Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Processamento Alternativo/genética , Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Éxons , Expressão Gênica , Humanos , NF-kappa B/metabolismo , Isoformas de Proteínas/genética , Precursores de RNA/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/genética
12.
Nucleic Acids Res ; 48(15): 8320-8331, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32749457

RESUMO

The rat is an important model organism in biomedical research for studying human disease mechanisms and treatments, but its annotated transcriptome is far from complete. We constructed a Rat Transcriptome Re-annotation named RTR using RNA-seq data from 320 samples in 11 different organs generated by the SEQC consortium. Totally, there are 52 807 genes and 114 152 transcripts in RTR. Transcribed regions and exons in RTR account for ∼42% and ∼6.5% of the genome, respectively. Of all 73 074 newly annotated transcripts in RTR, 34 213 were annotated as high confident coding transcripts and 24 728 as high confident long noncoding transcripts. Different tissues rather than different stages have a significant influence on the expression patterns of transcripts. We also found that 11 715 genes and 15 852 transcripts were expressed in all 11 tissues and that 849 house-keeping genes expressed different isoforms among tissues. This comprehensive transcriptome is freely available at http://www.unimd.org/rtr/. Our new rat transcriptome provides essential reference for genetics and gene expression studies in rat disease and toxicity models.


Assuntos
Genoma/genética , Anotação de Sequência Molecular , RNA-Seq/métodos , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ratos , Sequenciamento Completo do Exoma
13.
Nucleic Acids Res ; 48(15): 8724-8739, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32735645

RESUMO

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Motivated by our previous finding that intron retention (IR) could lead to transcript instability, in this study, we performed BruChase-Seq to experimentally monitor the expression dynamics of nascent transcripts in resting and activated CD4+ T cells. Computational modeling was then applied to quantify the stability of spliced and intron-retained transcripts on a genome-wide scale. Beyond substantiating that intron-retained transcripts were considerably less stable than spliced transcripts, we found a global stabilization of spliced mRNAs upon T cell activation, although the stability of intron-retained transcripts remained relatively constant. In addition, we identified that La-related protein 4 (LARP4), an RNA-binding protein (RBP) known to enhance mRNA stability, was involved in T cell activation-dependent mRNA stabilization. Knocking out Larp4 in mice destabilized Nfκb1 mRNAs and reduced secretion of interleukin-2 (IL2) and interferon-gamma (IFNγ), two factors critical for T cell proliferation and function. We propose that coordination between splicing regulation and mRNA stability may provide a novel paradigm to control spatiotemporal gene expression during T cell activation.


Assuntos
Interferon gama/genética , Interleucina-2/genética , Proteínas/genética , Estabilidade de RNA/genética , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Humanos , Íntrons/genética , Ativação Linfocitária/genética , Camundongos , NF-kappa B/genética , Ligação Proteica/genética , RNA Mensageiro/genética , Linfócitos T/metabolismo
14.
Nature ; 583(7818): 711-719, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32728246

RESUMO

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética , Processamento Alternativo/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Dados Genéticas , Feminino , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/genética , Masculino , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade por Substrato
15.
Gene ; 758: 144961, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32693148

RESUMO

Ginkgo biloba L. is regarded as the most ancient living tree, and its kernel has been used as a traditional Chinese medicine for more than 2,000 years. The leaf extracts of this tree have been among the bestselling herbal remedies in Western countries since the last century. To understand the biosynthesis of the pharmacologically active ingredients in G. biloba, flavonoids and terpenoid trilactones (TTLs), we sequenced the transcriptomes of G. biloba leaves, kernels and testae with Iso-Seq and RNA-Seq technologies and obtained 152,524 clean consensus reads. When these reads were used to improve the annotation of the G. biloba genome, 4,856 novel genes, 25,583 new isoforms of previously annotated genes and 4,363 lncRNAs were discovered. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that genes involved in growth, regulation and response to stress were more likely to be regulated by alternative splicing (AS) or alternative polyadenylation (APA), which represent the two most important posttranscriptional regulation mechanisms. It was found that some of the characterized genes involved in the biosynthesis of flavonoids and TTLs were also possibly regulated by AS and APA. Using phylogenetic and gene expression pattern analyses, some candidate genes for the biosynthesis of flavonoids and TTLs were screened. After qRT-PCR validation, the final candidate genes for flavonoid biosynthesis included three UDP-glycosyltransferases and one MYB transcription factor, while the candidate genes for TTL biosynthesis included two cytochrome P450 and one WRKY transcription factor. Our study suggested that Iso-Seq may play an important role in improving genome annotation, elucidating AS and APA mechanisms and discovering candidate genes involved in the biosynthesis of some secondary metabolites.


Assuntos
Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas/genética , Ginkgo biloba/genética , Ginkgo biloba/metabolismo , Lactonas/metabolismo , Terpenos/metabolismo , Processamento Alternativo/genética , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Genoma de Planta/genética , Extratos Vegetais , Transcriptoma , Sequenciamento Completo do Genoma
16.
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
17.
RNA ; 26(10): 1464-1480, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32631843

RESUMO

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.


Assuntos
Processamento Alternativo/genética , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Evolução Molecular , Duplicação Gênica/genética , Genoma/genética , Isoformas de Proteínas/genética
18.
Int J Hematol ; 112(3): 395-408, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32533515

RESUMO

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family members have cytidine deaminase activity and can induce cytosine to uracil transition in nucleic acid. The main function of APOBEC3 (A3) proteins is to trigger an innate immune response to viral infections. Recent reports have shown that several APOBEC family proteins such as A3B can induce somatic mutations into genomic DNA and thus promote cancer development. However, the role of A3D on somatic mutations is unclear. Here, we identified the alternative splicing of A3D, and investigated each splice variant's subcellular localization and role in DNA mutagenesis. We identified four A3D variants, which all have one or two cytidine deaminase domains. The full-length form of A3D (variant 1) and truncated forms of A3D (variant 2, 6, 7) showed the ability to induce C/G to T/A transitions in foreign DNA and genomic DNA and retained antiretroviral activity. Furthermore, we demonstrated that A3D and A3B could induce deletions that are possibly repaired by microhomology-mediated end joining (MMEJ). Taken together, our experiments illustrated that alternative splicing generates functional diversity of A3D, and some variants can act as DNA mutators in genomic DNA.


Assuntos
Processamento Alternativo/genética , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , DNA/genética , Mutação/genética , Antirretrovirais , Linhagem Celular Tumoral , Células Cultivadas , Citidina Desaminase/química , Reparo do DNA por Junção de Extremidades/genética , Variação Genética , Humanos , Domínios Proteicos
19.
Nature ; 582(7812): 438-442, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555469

RESUMO

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.


Assuntos
Processamento Alternativo/genética , Regulação Viral da Expressão Gênica , HIV-1/genética , Mutação , Sítios de Splice de RNA/genética , RNA Viral/química , RNA Viral/genética , Algoritmos , Sequência de Bases , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Dobramento de RNA , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Ésteres do Ácido Sulfúrico , Termodinâmica
20.
RNA ; 26(10): 1389-1399, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522889

RESUMO

Alternative splicing is responsible for much of the transcriptomic and proteomic diversity observed in eukaryotes and involves combinatorial regulation by many cis-acting elements and trans-acting factors. SR and hnRNP splicing regulatory proteins often have opposing effects on splicing efficiency depending on where they bind the pre-mRNA relative to the splice site. Position-dependent splicing repression occurs at spliceosomal E-complex, suggesting that U1 snRNP binds but cannot facilitate higher order spliceosomal assembly. To test the hypothesis that the structure of U1 snRNA changes during activation or repression, we developed a method to structure-probe native U1 snRNP in enriched conformations that mimic activated or repressed spliceosomal E-complexes. While the core of U1 snRNA is highly structured, the 5' end of U1 snRNA shows different SHAPE reactivities and psoralen crosslinking efficiencies depending on where splicing regulatory elements are located relative to the 5' splice site. A motif within the 5' splice site binding region of U1 snRNA is more reactive toward SHAPE electrophiles when repressors are bound, suggesting U1 snRNA is bound, but less base-paired. These observations demonstrate that splicing regulators modulate splice site selection allosterically.


Assuntos
Regulação Alostérica/genética , Processamento Alternativo/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Spliceossomos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteômica/métodos , Precursores de RNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética
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