RESUMO
The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.
Assuntos
Íntrons , Splicing de RNA , Spliceossomos , Humanos , Íntrons/genética , Spliceossomos/metabolismo , Células HEK293 , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/genética , Éxons/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Células HeLa , Sítios de Splice de RNARESUMO
BACKGROUND: The removal of introns occurs through the splicing of a 5' splice site (5'ss) with a 3' splice site (3'ss). These two elements are recognized by distinct components of the spliceosome. However, introns in higher eukaryotes contain many matches to the 5' and 3' splice-site motifs that are presumed not to be used. RESULTS: Here, we find that many of these sites can be used. We also find occurrences of the AGGT motif that can function as either a 5'ss or a 3'ss-previously referred to as dual-specific splice sites (DSSs)-within introns. Analysis of the Sequence Read Archive reveals a 3.1-fold enrichment of DSSs relative to expectation, implying synergy between the ability to function as a 5'ss and 3'ss. Despite this suggested mechanistic advantage, DSSs are 2.7- and 4.7-fold underrepresented in annotated 5' and 3' splice sites. A curious exception is the polyubiquitin gene UBC, which contains a tandem array of DSSs that precisely delimit the boundary of each ubiquitin monomer. The resulting isoforms splice stochastically to include a variable number of ubiquitin monomers. We found no evidence of tissue-specific or feedback regulation but note the 8.4-fold enrichment of DSS-spliced introns in tandem repeat genes suggests a driving role in the evolution of genes like UBC. CONCLUSIONS: We find an excess of unannotated splice sites and the utilization of DSSs in tandem repeats supports the role of splicing in gene evolution. These findings enhance our understanding of the diverse and complex nature of the splicing process.
Assuntos
Poliubiquitina , Splicing de RNA , Poliubiquitina/genética , Íntrons , Sítios de Splice de RNA , ArquivosRESUMO
The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of the first viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectroscopy. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.
RESUMO
Humans coexisted and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here, we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,169 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 962 exonic splicing mutations that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than that in Neanderthals. Adaptively introgressed variants were enriched for moderate-effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly compelling examples, we characterized a unique tissue-specific alternative splicing variant at the adaptively introgressed innate immunity gene TLR1, as well as a unique Neanderthal introgressed alternative splicing variant in the gene HSPG2 that encodes perlecan. We further identified potentially pathogenic splicing variants found only in Neanderthals and Denisovans in genes related to sperm maturation and immunity. Finally, we found splicing variants that may contribute to variation among modern humans in total bilirubin, balding, hemoglobin levels, and lung capacity. Our findings provide unique insights into natural selection acting on splicing in human evolution and demonstrate how functional assays can be used to identify candidate causal variants underlying differences in gene regulation and phenotype.
Assuntos
Hominidae , Homem de Neandertal , Masculino , Animais , Humanos , Homem de Neandertal/genética , Sêmen , Hominidae/genética , Alelos , Regulação da Expressão Gênica , Genoma HumanoRESUMO
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is transmitted through airborne particles in exhaled breath, causing severe respiratory disease, coronavirus disease-2019 (COVID-19), in some patients. Samples for SARS-CoV-2 testing are typically collected by nasopharyngeal swab, with the virus detected by PCR; however, patients can test positive for 3 months after infection. Without the capacity to assay SARS-CoV-2 in breath, it is not possible to understand the risk for transmission from infected individuals. To detect virus in breath, the Bubbler-a breathalyzer that reverse-transcribes RNA from SARS-CoV-2 particles into a sample-specific barcoded cDNA-was developed. In a study of 70 hospitalized patients, the Bubbler was both more predictive of lower respiratory tract involvement (abnormal chest X-ray) and less invasive than alternatives. Samples tested using the Bubbler were threefold more enriched for SARS-CoV-2 RNA than were samples from tongue swabs, implying that virus particles were being directly sampled. The barcode-enabled Bubbler was used for simultaneous diagnosis in large batches of pooled samples at a lower limit of detection of 334 genomic copies per sample. Diagnosis by sequencing can provide additional information, such as viral load and strain identity. The Bubbler was configured to sample nucleic acids in water droplets circulating in air, demonstrating its potential in environmental monitoring and the protective effect of adequate ventilation.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Testes Diagnósticos de Rotina/métodos , Sistema Respiratório/virologia , SARS-CoV-2/genética , Líquidos Corporais/virologia , COVID-19/virologia , Humanos , RNA Viral/genética , Manejo de Espécimes , Carga Viral/métodosRESUMO
BACKGROUND: Mutation rates vary across the genome. Many trans factors that influence mutation rates have been identified, as have specific sequence motifs at the 1-7-bp scale, but cis elements remain poorly characterized. The lack of understanding regarding why different sequences have different mutation rates hampers our ability to identify positive selection in evolution and to identify driver mutations in tumorigenesis. RESULTS: Here, we use a combination of synthetic genes and sequences of thousands of isolated yeast colonies to show that intrinsic DNA curvature is a major cis determinant of mutation rate. Mutation rate negatively correlates with DNA curvature within genes, and a 10% decrease in curvature results in a 70% increase in mutation rate. Consistently, both yeast and humans accumulate mutations in regions with small curvature. We further show that this effect is due to differences in the intrinsic mutation rate, likely due to differences in mutagen sensitivity and not due to differences in the local activity of DNA repair. CONCLUSIONS: Our study establishes a framework for understanding the cis properties of DNA sequence in modulating the local mutation rate and identifies a novel causal source of non-uniform mutation rates across the genome.
Assuntos
DNA/química , Taxa de Mutação , Carcinogênese/genética , Reparo de Erro de Pareamento de DNA , Evolução Molecular , Genômica , Humanos , Mutagênicos/toxicidade , Neoplasias/genética , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cucumber (Cucumis sativus L.), a kind of fruit usually harvested at the immature green stage, belongs to non-climacteric fruit. To investigate the contribution of abscisic acid (ABA) to cucumber fruit development and ripening, variation in ABA level was investigated and a peak in ABA level was found in pulp before fruit get fully ripe. To clarify this point further, exogenous ABA was applied to cucumber fruits at two different development stages. Results showed that ABA application at the turning stage promotes cucumber fruit ripening, while application at the immature green stage had inconspicuous effects. In addition, with the purpose of understanding the transcriptional regulation of ABA, two partial cDNAs of CsNCED1 and CsNCED2 encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthetic pathway; one partial cDNA of CsCYP707A1 for 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA and two partial cDNAs of CsBG1 and CsBG2 for ß-glucosidase (BG) that hydrolyzes ABA glucose ester (ABA-GE) to release active ABA were cloned from cucumber. The DNA and deduced amino acid sequences of these obtained genes respectively showed high similarities to their homologous genes in other plants. Real-time PCR analysis revealed that ABA content may be regulated by its biosynthesis (CsNCEDs), catabolism (CsCYP707A1) and reactivation genes (CsBGs) at the transcriptional level during cucumber fruit development and ripening, in response to ABA application, dehydration and pollination, among which CsNCED1, CsCYP707A1 and CsBG1 were highly expressed in pulp and may play more important roles in regulating ABA metabolism.
Assuntos
Ácido Abscísico/metabolismo , Cucumis/metabolismo , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Plantas/genética , Transcrição Gênica , Ácido Abscísico/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cucumis/enzimologia , Cucumis/genética , Cucumis/crescimento & desenvolvimento , DNA Complementar , DNA de Plantas , Desidratação , Frutas/enzimologia , Frutas/crescimento & desenvolvimento , Dados de Sequência Molecular , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Polinização , Homologia de SequênciaRESUMO
The abscisic acid (ABA) signal transduction core components in plants include the ABA receptors (PYR/PYL/RCARs), the group A type 2C protein phosphatases (PP2Cs) and the subclass III SNF1-related protein kinases 2 (SnRK2s). In this study, via homology cloning, three CsPYLs, four CsPP2Cs and two CsSnRK2s partial cDNAs were obtained in cucumber (Cucumis sativus). In silico analysis results indicated that all CsPYL, CsPP2C, and CsSnRK2 genes obtained are homologous to Arabidopsis AtPYL, AtPP2C, and AtSnRK2 genes, respectively. Based on phylogenetic analysis, only CsPP2C2 and CsSnRK2.2 belonged to group A PP2C and subclass III SnRK2, respectively, and may involve in ABA signal transduction. During cucumber fruit development and ripening, CsPYL2 and CsPP2C2 were highly expressed and both reached their peak value at 27 DAF, at which stage the ABA content was also at its highest level, which indicated that the CsPYL2 and CsPP2C2 may involve in transducing ABA signal in fruit and regulating fruit development and ripening. Under drought stress condition in cucumber seedlings, CsPYL1, CsPYL2, CsPP2C2 and CsSnKR2.2 were sensitive and up-regulated in root, stem and leaf; meanwhile, CsPYL3 showed a low sensitivity and were down-regulated in root and stem.
Assuntos
Ácido Abscísico/metabolismo , Cucumis sativus/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Ácido Abscísico/análise , Biomassa , Carboidratos/análise , Clorofila/metabolismo , Cucumis sativus/crescimento & desenvolvimento , Cucumis sativus/metabolismo , Regulação para Baixo , Secas , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transdução de Sinais , Estresse Fisiológico , Regulação para CimaRESUMO
To study the role of ABA in development and ripening of strawberry fruit, two ABA key synthetase genes FaNCED1, FaNCED2 and one ABA key degradation enzyme gene FaCYP707A1 were cloned from strawberry cultivar 'Ablion'. The three genes and putative amino acid sequences, respectively, had high similarities with their homologues in other plants. In strawberry pulp, expression of FaNCED2 rose in two stages that corresponded with increases in ABA levels. The expression of FaNCED1 was much lower and increased continually. The expression of FaCYP707A1 increased as fruit changed from bright green to white, then decreased as it ripened. Auxin reduced expression of these three genes. Analysis of expression of these genes in different organs and tissues showed that FaNCED2 was abundant in mature achenes and the pulp (receptacle) had high expression of FaNCED1 and FaCYP707A1. ABA may play a regulation role in strawberry fruit development and ripening. The content of ABA was regulated by its key synthetase gene NCED2 and degradation gene CYP707A1.
RESUMO
The aim of this study was to obtain new insights into the mechanisms that regulate endogenous abscisic acid (ABA) levels by ß-glucosidase genes during the development of watermelons (Citrullus lanatus) and under drought stress conditions. In total, five cDNAs from watermelons were cloned by using reverse transcription-PCR (RT-PCR). They included three cDNAs (ClBG1, ClBG2 and ClBG3) homologous to those that encode ß-glucosidase l that hydrolyzes the ABA glucose ester (ABA-GE) to release active ABA, ClNCED4, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, and ClCYP707A1, encoding ABA 8'-hydroxylase. A BLAST homology search revealed that the sequences of cDNAs and the deduced amino acids of these genes showed a high degree of homology to comparable molecules of other plant species. During fruit development and ripening, the expressions of ClBG1, ClNCED4 and ClCYP707A1 were relatively low at an early stage, increased rapidly along with fruit ripening, and reached the highest levels at 27 days after full bloom (DAFB) at the harvest stage. This trend was consistent with the accumulation of ABA. The ClBG2 gene on the other hand was highly expressed at 5 DAFB, and then decreased gradually with fruit development. Unlike ClBG1 and ClBG2, the expression of ClBG3 was low at an early stage; its expression peak occurred at 15 DAFB and then declined to the lowest point. When watermelon seedlings were subjected to drought stress, expressions of ClBG1 and ClCYP707A1 were significantly down-regulated, while expressions of ClBG2 and ClNCED4 were up-regulated in the roots, stems and leaves. The expression of ClBG3 was down-regulated in root tissue, but was up-regulated in stems and leaves. In conclusion, endogenous ABA content was modulated by a dynamic balance between biosynthesis and catabolism regulated by ClNCED4, ClCYP707A1 and ClBGs during development and under drought stress condition. It seems likely that ß-glucosidase genes are important for this regulation process.
Assuntos
Ácido Abscísico/metabolismo , Citrullus/enzimologia , Frutas/enzimologia , Homeostase/fisiologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , beta-Glucosidase/metabolismo , Citrullus/genética , Citrullus/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/análise , DNA Complementar/genética , DNA de Plantas/análise , DNA de Plantas/genética , Desidratação/genética , Desidratação/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Secas , Frutas/genética , Frutas/fisiologia , Regulação da Expressão Gênica , Filogenia , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Glucosidase/genéticaRESUMO
Cell wall catabolism during fruit ripening is under complex control and is key for fruit quality and shelf life. To examine the role of abscisic acid (ABA) in tomato (Solanum lycopersicum) fruit ripening, we suppressed SlNCED1, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of ABA. To suppress SlNCED1 specifically in tomato fruits, and thus avoid the pleiotropic phenotypes associated with ABA deficiency, we used an RNA interference construct driven by the fruit-specific E8 promoter. ABA accumulation and SlNCED1 transcript levels in the transgenic fruit were down-regulated to between 20% and 50% of the levels measured in the control fruit. This significant reduction in NCED activity led to a down-regulation in the transcription of genes encoding major cell wall catabolic enzymes, specifically polygalacturonase (SlPG), pectin methyl esterase (SlPME), ß-galactosidase precursor mRNA (SlTBG), xyloglucan endotransglycosylase (SlXET), endo-1,4-ß-cellulose (SlCels), and expansin (SlExp). This resulted in an increased accumulation of pectin during ripening. In turn, this led to a significant extension of the shelf life to 15 to 29 d compared with a shelf life of only 7 d for the control fruit and an enhancement of fruit firmness at the mature stage by 30% to 45%. In conclusion, ABA affects cell wall catabolism during tomato fruit ripening via down-regulation of the expression of major catabolic genes (SlPG, SlPME, SlTBG, SlXET, SlCels, and SlExp).