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1.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 42(3): 289-296, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32616122

RESUMO

Objective To analyze the expression of splicing factors in gastric cancer using bioinformatics methods and investigate the effect of aberrantly expressed serine/arginine-rich splicing factor(SRSF10)on the phenotype of gastric cancer cells. Methods The RNA-seq data of gastric cancer and paracancerous tissues were downloaded from The Cancer Genome Atlas(TCGA)cancer database,and bioinformatics analysis was performed to obtain the splicing factors differentially expressed in gastric cancer.The splicing factor SRSF10 was selected to investigate its effect on the development of gastric cancer.RNA interference technology was used to construct SRSF10 knockdown gastric cancer cells.MTS,Transwell,and cell scratches were used to study the effect of SRSF10 knockdown on gastric cancer cell phenotype. Results A total of 48 splicing factors were identified in gastric cancer by a series of bioinformatics techniques,of which 35 were up-regulated and 13 were down-regulated.The splicing factor SRSF10,which was up-regulated,was selected for further study.It was found that the gastric cancer cells after SRSF10 knockdown proliferated more slowly and had lower migration ability than normal gastric cancer cells. Conclusions Multiple splicing factors are found in gastric cancer and may play an important role in the development of gastric cancer.The splicing factor SRSF10 may contribute to the pathogenesis of gastric cancer.


Assuntos
Biologia Computacional , Neoplasias Gástricas , Processamento Alternativo , Proteínas de Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Processamento de RNA , Proteínas Repressoras , Fatores de Processamento de Serina-Arginina
2.
PLoS Biol ; 18(6): e3000722, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569301

RESUMO

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.


Assuntos
Macrófagos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Processamento Alternativo/genética , Animais , Cálcio/metabolismo , Espaço Extracelular/metabolismo , Inativação Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Íons , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Células RAW 264.7 , Cloreto de Sódio/farmacologia
3.
Mol Genet Genomics ; 295(5): 1227-1237, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32524299

RESUMO

The silk gland of the silkworm Bombyx mori is a specialized organ where silk proteins are efficiently synthesized under precise regulation that largely determines the properties of silk fibers. To understand the genes involved in the regulation of silk protein synthesis, considerable research has focused on the transcripts expressed in silk glands; however, the complete transcriptome profile of this organ has yet to be elucidated. Here, we report a full-length silk gland transcriptome obtained by PacBio single-molecule long-read sequencing technology. In total, 11,697 non-redundant transcripts were identified in mixed samples of silk glands dissected from larvae at five developmental stages. When compared with the published reference, the full-length transcripts optimized the structures of 3002 known genes, and a total of 9061 novel transcripts with an average length of 2171 bp were detected. Among these, 1403 (15.5%) novel transcripts were computationally revealed to be lncRNAs, 8135 (89.8%) novel transcripts were annotated to different protein and nucleotide databases, and 5655 (62.4%) novel transcripts were predicted to have complete ORFs. Furthermore, we found 1867 alternative splicing events, 2529 alternative polyadenylation events, 784 fusion events and 6596 SSRs. This study provides a comprehensive set of reference transcripts and greatly revises and expands the available silkworm transcript data. In addition, these data will be very useful for studying the regulatory mechanisms of silk protein synthesis.


Assuntos
Bombyx/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Seda/genética , Imagem Individual de Molécula/métodos , Processamento Alternativo , Animais , Bombyx/genética , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/genética , Fases de Leitura Aberta , Poliadenilação , RNA Longo não Codificante/genética , Sequenciamento Completo do Exoma
4.
DNA Cell Biol ; 39(7): 1243-1255, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32543226

RESUMO

Increasing evidence have demonstrated that dysregulated alternative splicing (AS) events promoted tumor development and was correlated with worse prognosis in the context of certain malignancies. Nevertheless, a comprehensive examination of the prognosis role of AS events in acute myeloid leukemia (AML) has not yet been illuminated. In this study, univariate and multivariate Cox regression analysis were used to identify survival-related AS events and independent prognostic predictors. The interaction between splicing factors (SFs) and AS events was visualized by Cytoscape. A total of 3013 survival-associated AS events in 1977 genes were screened in 151 AML patients. Interestingly, the majority (2031 events) were revealed to be protective factors. Furthermore, the prediction models were constructed for each type of AS and all of them displayed good performance in predicting prognosis, considering their area under curve values of the receiver operating characteristic were all above 0.7. Notably, the splicing regulatory network displayed the underlying interaction networks between SFs and AS events. Taken together, our study demonstrated the survival-related AS events in AML and uncovered the possible association between SFs and prognostic AS events, which provide new prognostic biomarkers and aid to develop novel targets for AML therapy.


Assuntos
Processamento Alternativo , Biologia Computacional , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Humanos , Prognóstico
5.
PLoS Genet ; 16(5): e1008804, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32407316

RESUMO

Cattle are ideally suited to investigate the genetics of male reproduction, because semen quality and fertility are recorded for all ejaculates of artificial insemination bulls. We analysed 26,090 ejaculates of 794 Brown Swiss bulls to assess ejaculate volume, sperm concentration, sperm motility, sperm head and tail anomalies and insemination success. The heritability of the six semen traits was between 0 and 0.26. Genome-wide association testing on 607,511 SNPs revealed a QTL on bovine chromosome 6 that was associated with sperm motility (P = 2.5 x 10-27), head (P = 2.0 x 10-44) and tail anomalies (P = 7.2 x 10-49) and insemination success (P = 9.9 x 10-13). The QTL harbors a recessive allele that compromises semen quality and male fertility. We replicated the effect of the QTL on fertility (P = 7.1 x 10-32) in an independent cohort of 2481 Brown Swiss bulls. The analysis of whole-genome sequencing data revealed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 encoding WD repeat-containing protein 19 was in linkage disequilibrium with the fertility-associated haplotype. WD repeat-containing protein 19 is a constituent of the intraflagellar transport complex that is essential for the physiological function of motile cilia and flagella. Bioinformatic and transcription analyses revealed that the BTA6:58373887 T-allele activates a cryptic exonic splice site that eliminates three evolutionarily conserved amino acids from WDR19. Western blot analysis demonstrated that the BTA6:58373887 T-allele decreases protein expression. We make the remarkable observation that, in spite of negative effects on semen quality and bull fertility, the BTA6:58373887 T-allele has a frequency of 24% in the Brown Swiss population. Our findings are the first to uncover a variant that is associated with quantitative variation in semen quality and male fertility in cattle.


Assuntos
Processamento Alternativo , Proteínas do Citoesqueleto/genética , Infertilidade Masculina/genética , Polimorfismo de Nucleotídeo Único , Sêmen/fisiologia , Animais , Bovinos , Cromossomos de Mamíferos/genética , Estudo de Associação Genômica Ampla , Inseminação Artificial/veterinária , Masculino , Característica Quantitativa Herdável , Análise do Sêmen/veterinária , Motilidade Espermática , Sequenciamento Completo do Genoma
6.
PLoS Genet ; 16(5): e1008782, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32421721

RESUMO

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ventrículos do Coração/citologia , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Processamento Alternativo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Contagem de Células , Células Cultivadas , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
8.
Nat Commun ; 11(1): 2140, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358586

RESUMO

The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Sinapses/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Sítios de Ligação/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/fisiologia
9.
Nat Commun ; 11(1): 2089, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350277

RESUMO

The role of dysregulation of mRNA alternative splicing (AS) in the development and progression of solid tumors remains to be defined. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) evolution. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent genomic copy number variations (CNVs), leading to mis-expression of ~68% of SRGs during PCa development and progression. Consequently, many SRGs are prognostic. Surprisingly, androgen receptor controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and the spliceosome as a therapeutic vulnerability for CRPC.


Assuntos
Íntrons/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Spliceossomos/metabolismo , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Estudos de Coortes , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Compostos de Epóxi/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Macrolídeos/farmacologia , Masculino , Camundongos , Terapia de Alvo Molecular , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Transcrição Genética/efeitos dos fármacos
10.
Clin Sci (Lond) ; 134(10): 1143-1150, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32442315

RESUMO

Angiotensin-converting enzyme 2 (ACE2) plays an essential role in maintaining the balance of the renin-angiotensin system and also serves as a receptor for the SARS-CoV-2, SARS-CoV, and HCoV-NL63. Following the recent outbreak of SARS-CoV-2 infection, there has been an urgent need to develop therapeutic interventions. ACE2 is a potential target for many treatment approaches for the SARS-CoV-2. With the help of bioinformatics, we have predicted several novel exons of the human ACE2 gene. The inclusion of novel exons located in the 5'UTR/intronic region in the mature transcript may remove the critical ACE2 residues responsible for the interaction with the receptor-binding domain (RBD) of SARS-CoV-2, thus preventing their binding and entry into the cell. Additionally, inclusion of a novel predicted exons located in the 3'UTR by alternative splicing may remove the C-terminal transmembrane domain of ACE2 and generate soluble ACE2 isoforms. Splice-switching antisense oligonucleotides (SSOs) have been employed effectively as a therapeutic strategy in several disease conditions. Alternative splicing of the ACE2 gene could similarly be modulated using SSOs to exclude critical domains required for the entry of SARS-CoV-2. Strategies can also be designed to deliver these SSOs directly to the lungs in order to minimize the damage caused by SARS-CoV-2 pathogenesis.


Assuntos
Processamento Alternativo , Infecções por Coronavirus/genética , Oligonucleotídeos Antissenso/farmacologia , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Internalização do Vírus , Betacoronavirus/fisiologia , Biologia Computacional , Infecções por Coronavirus/terapia , Éxons , Humanos , Modelos Moleculares , Pandemias , Pneumonia Viral/terapia , Domínios Proteicos , Receptores Virais/genética
11.
DNA Cell Biol ; 39(6): 1051-1063, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32379494

RESUMO

Triple-negative breast cancer (TNBC) is a high-risk subtype of breast cancer defined by negative expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Accumulating evidence indicates that alternative splicing (AS) events are correlated with the prognosis of cancer. RNA sequencing data and AS event data were manually curated from The Cancer Genome Atlas (TCGA) dataset and TCGA Splice Seq, respectively. Univariate and multivariate Cox regression analyses were applied to screen AS events associated with TNBC survival and to establish a prognostic model. A receiver operating characteristic (ROC) curve was used to evaluate the performance of the prognostic model. Differentially expressed gene analysis and functional enrichment analysis were harnessed to reveal the functional role of gene sets and to screen novel biomarkers. By integrated bioinformatics analysis of AS events and gene expression in TNBC, our study is the first to generate specific AS event profiles, prognostic AS event interaction networks, and splice factor-AS interaction networks for TNBC. Surprisingly, we found that the performance of the AS-based prognostic model was encouraging with a mean area under the ROC curve of 0.957 at 2-10 years. We also found that chemokine (C-C motif) ligand 16 (CCL16) expression was correlated with TNBC grade and could be a potential novel biomarker. In conclusion, this study provided a systematic analysis of prognostic AS event profiles and gene expression in TNBC. A novel prognostic model based on AS events may establish a foundation for future research investigating the diagnosis and treatment of TNBC.


Assuntos
Processamento Alternativo , Neoplasias de Mama Triplo Negativas/genética , Marcadores Genéticos/genética , Humanos , Prognóstico , RNA Mensageiro/genética , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/diagnóstico
12.
PLoS Pathog ; 16(4): e1008401, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32302366

RESUMO

Alternative splicing (AS) of pre-mRNAs in plants is an important mechanism of gene regulation in environmental stress tolerance but plant signals involved are essentially unknown. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is mediated by mitogen-activated protein kinases and the majority of PTI defense genes are regulated by MPK3, MPK4 and MPK6. These responses have been mainly analyzed at the transcriptional level, however many splicing factors are direct targets of MAPKs. Here, we studied alternative splicing induced by the PAMP flagellin in Arabidopsis. We identified 506 PAMP-induced differentially alternatively spliced (DAS) genes. Importantly, of the 506 PAMP-induced DAS genes, only 89 overlap with the set of 1950 PAMP-induced differentially expressed genes (DEG), indicating that transcriptome analysis does not identify most DAS events. Global DAS analysis of mpk3, mpk4, and mpk6 mutants in the absence of PAMP treatment showed no major splicing changes. However, in contrast to MPK3 and MPK6, MPK4 was found to be a key regulator of PAMP-induced DAS events as the AS of a number of splicing factors and immunity-related protein kinases is affected, such as the calcium-dependent protein kinase CPK28, the cysteine-rich receptor like kinases CRK13 and CRK29 or the FLS2 co-receptor SERK4/BKK1. Although MPK4 is guarded by SUMM2 and consequently, the mpk4 dwarf and DEG phenotypes are suppressed in mpk4 summ2 mutants, MPK4-dependent DAS is not suppressed by SUMM2, supporting the notion that PAMP-triggered MPK4 activation mediates regulation of alternative splicing.


Assuntos
Processamento Alternativo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/genética , Padrões Moleculares Associados a Patógenos/metabolismo , Imunidade Vegetal/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Flagelina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Fisiológico
13.
PLoS One ; 15(4): e0231000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287265

RESUMO

Myotonic dystrophy type 1 (DM1) is a rare genetic disorder, characterised by muscular dystrophy, myotonia, and other symptoms. DM1 is caused by the expansion of a CTG repeat in the 3'-untranslated region of DMPK. Longer CTG expansions are associated with greater symptom severity and earlier age at onset. The primary mechanism of pathogenesis is thought to be mediated by a gain of function of the CUG-containing RNA, that leads to trans-dysregulation of RNA metabolism of many other genes. Specifically, the alternative splicing (AS) and alternative polyadenylation (APA) of many genes is known to be disrupted. In the context of clinical trials of emerging DM1 treatments, it is important to be able to objectively quantify treatment efficacy at the level of molecular biomarkers. We show how previously described candidate mRNA biomarkers can be used to model an effective reduction in CTG length, using modern high-dimensional statistics (machine learning), and a blood and muscle mRNA microarray dataset. We show how this model could be used to detect treatment effects in the context of a clinical trial.


Assuntos
Distrofia Miotônica/genética , Distrofia Miotônica/terapia , RNA Mensageiro/genética , Processamento Alternativo , Bioestatística , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/estatística & dados numéricos , Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Marcadores Genéticos , Humanos , Análise dos Mínimos Quadrados , Aprendizado de Máquina , Modelos Genéticos , Músculos/metabolismo , Distrofia Miotônica/metabolismo , Miotonina Proteína Quinase/genética , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Poliadenilação , RNA Mensageiro/metabolismo , Resultado do Tratamento , Expansão das Repetições de Trinucleotídeos
14.
Gene ; 749: 144708, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32334022

RESUMO

The kallikrein-related peptidase 15 (KLK15) gene is a member of the largest cluster of serine proteases in the human genome. Exhibiting trypsin-like activity, KLK15 is most likely involved in the activation of prostate-specific antigen (PSA; also known as KLK3), an established biomarker for the diagnosis and screening of prostate cancer. High mRNA expression levels of KLK15 have already been reported in ovarian and prostate cancer, in contrast with breast cancer, where KLK15 has been proposed as a biomarker of favorable prognosis. In this study, we exploited the next-generation sequencing (NGS) technology along with 3' rapid amplification of cDNA ends (3' RACE) to discover alternative KLK15 splice variants. Extensive computational analysis of the obtained NGS data revealed the existence of novel splice junctions, thus supporting the existence of novel KLK15 transcripts. Six novel KLK15 splice variants were identified and verified by Sanger sequencing. Two of them (KLK15 v.11 and v.12) contain an open reading frame and are hence predicted to encode two novel KLK15 protein isoforms. Expression analysis of each KLK15 splice variant in sixteen cDNA pools from malignant cell lines and in normal cell lines (HEK293, HaCaT, and BJ cells) revealed very different expression profiles of particular KLK15 transcripts. Moreover, the new KLK15 splice variants were shown to be expressed in breast, ovarian, prostate, urinary bladder, colon, and renal tissue specimens. Due to the prominent clinical value of KLK15 mRNA expression, the novel KLK15 transcripts appear as candidate cancer biomarkers for diagnostic and/or prognostic purposes and, therefore, merit further investigation.


Assuntos
Processamento Alternativo , Calicreínas/genética , Linhagem Celular , Linhagem Celular Tumoral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Calicreínas/metabolismo
15.
PLoS Genet ; 16(4): e1008662, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32310939

RESUMO

African Americans (AAs) are disproportionately affected by metabolic diseases and adverse drug events, with limited publicly available genomic and transcriptomic data to advance the knowledge of the molecular underpinnings or genetic associations to these diseases or drug response phenotypes. To fill this gap, we obtained 60 primary hepatocyte cultures from AA liver donors for genome-wide mapping of expression quantitative trait loci (eQTL) using LAMatrix. We identified 277 eGenes and 19,770 eQTLs, of which 67 eGenes and 7,415 eQTLs are not observed in the Genotype-Tissue Expression Project (GTEx) liver eQTL analysis. Of the eGenes found in GTEx only 25 share the same lead eQTL. These AA-specific eQTLs are less correlated to GTEx eQTLs. in effect sizes and have larger Fst values compared to eQTLs found in both cohorts (overlapping eQTLs). We assessed the overlap between GWAS variants and their tagging variants with AA hepatocyte eQTLs and demonstrated that AA hepatocyte eQTLs can decrease the number of potential causal variants at GWAS loci. Additionally, we identified 75,002 exon QTLs of which 48.8% are not eQTLs in AA hepatocytes. Our analysis provides the first comprehensive characterization of AA hepatocyte eQTLs and highlights the unique discoveries that are made possible due to the increased genetic diversity within the African ancestry genome.


Assuntos
Afro-Americanos/genética , Expressão Gênica/genética , Hepatócitos/metabolismo , Locos de Características Quantitativas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo/genética , Citocromo P-450 CYP3A/genética , Éxons/genética , Feminino , Predisposição Genética para Doença , Genética Médica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Fígado/citologia , Masculino , Proteínas do Tecido Nervoso/genética , Medicina de Precisão
16.
Nat Commun ; 11(1): 1768, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286305

RESUMO

The differential production of transcript isoforms from gene loci is a key cellular mechanism. Yet, its impact in protein production remains an open question. Here, we describe ORQAS (ORF quantification pipeline for alternative splicing), a pipeline for the translation quantification of individual transcript isoforms using ribosome-protected mRNA fragments (ribosome profiling). We find evidence of translation for 40-50% of the expressed isoforms in human and mouse, with 53% of the expressed genes having more than one translated isoform in human, and 33% in mouse. Differential splicing analysis revealed that about 40% of the splicing changes at RNA level are concordant with changes in translation. Furthermore, orthologous cassette exons between human and mouse preserve the directionality of the change, and are enriched in microexons in a comparison between glia and glioma. ORQAS leverages ribosome profiling to uncover a widespread and evolutionarily conserved impact of differential splicing on translation, particularly of microexon-containing isoforms.


Assuntos
Proteoma/metabolismo , Processamento de RNA/fisiologia , Processamento Alternativo/genética , Animais , Biologia Computacional/métodos , Humanos , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/genética , Processamento de RNA/genética , Ribossomos/genética , Ribossomos/metabolismo , Transcriptoma/genética
17.
Nat Commun ; 11(1): 1674, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245946

RESUMO

Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.


Assuntos
Proteínas CELF1/metabolismo , Proteína Semelhante a ELAV 4/genética , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/crescimento & desenvolvimento , Neurogênese/genética , Regiões 5' não Traduzidas/genética , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Feminino , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neocórtex/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Polirribossomos/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas/genética , Isoformas de RNA/genética , RNA-Seq
18.
Gene ; 746: 144659, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32276000

RESUMO

The genetic polymorphism of cytochrome P450 (CYPs)drug-metabolizing enzymes are well studied in human populations for drug safety and efficacy. CYP2C9 is a highly polymorphic CYP enzyme that oxidizing the indigenous compounds and xenobiotics. The present study was pursued to evaluate the genetic variation across the CYP2C9 gene among major groups of the Pakistani population. The CYP2C9 genomic region holding important warfarin drug-metabolizing SNPs was sequenced from 159 individuals belong from five major ethnic groups of Pakistani population. The population genetic analyses of the high-quality sequences data was performed using Arlequin v3.5, DnaSP v6.12 and Network 5 resources. The data analyses unveiled that genetic variance among samples mainly arose from population-scale differentiation among these ethnic groups with global Fst of 0.78, P-value < 0.0001. The highest pairwise population genetic variation observed between Saraiki and Baloch groups based on different statistical tests. Whereas, uniform genetic composition across CYP2C9 loci was inferred among Punjabi, Pathan and Sindhi groups with minimal genetic differentiation. Several SNPs, including the previously reported warfarin associated variants, i.e. rs2860905, rs1799853 (CYP2C9*2) and rs72558189 (CYP2C9*14) were detected in these population groups with diverse allelic frequencies. Besides, a novel intronic SNP, i.e. not available in dbSNP and Ensemble databases, was identified for a Sindhi individual sample. This novel SNP predicted to influence the CYP2C9 alternative transcript splicing. The pharmacogeneticsassessment of the CYP2C9 genetic variations identified in current study may important to test against the warfarin efficacy for different ethnicity of Pakistani population.


Assuntos
Processamento Alternativo , Citocromo P-450 CYP2C9/genética , Grupos Étnicos/genética , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Citocromo P-450 CYP2C9/metabolismo , Feminino , Humanos , Masculino , Paquistão/etnologia , Varfarina/farmacocinética , Varfarina/uso terapêutico
19.
Anticancer Res ; 40(4): 2033-2042, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234894

RESUMO

BACKGROUND: SF3B4, a critical component of U2 pre-mRNA spliceosomal complex, has been recently indicated as a potential oncogene in hepatocellular carcinoma (HCC). However, limited information exists on how SF3B4 expression is regulated in HCC. MATERIALS AND METHODS: To determine the regulatory factor for SF3B4 expression, small interfering RNA (siRNA), real-time polymerase chain reaction (qRT-PCR) and western blotting assay were performed. The in vivo expression profiles of SRSF3 and SF3B4 were analyzed using public datasets and clinical samples. RESULTS: Among 10 liver-specific splicing factors, only SRSF3 knockdown resulted in a significant increase in SF3B4 mRNA and consequently protein levels in SNU-368 HCC cells, probably via the retardation of SF3B4 mRNA decay rates. Using green fluorescent protein-SF3B4 fusion construct, the coding region of SF3B4 was found to be involved in SRSF3-mediated regulation of SF3B4 expression. Publicly available data from paired normal and tumor tissues in HCC and results from patients with HCC suggest that SRSF3 and SF3B4 possess an inverse relationship. CONCLUSION: SRSF3 is a key molecule for determining SF3B4 levels in HCC cells.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
20.
Gene ; 747: 144684, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32311412

RESUMO

PRMT8 is a neuron-specific protein arginine methyltransferase in vertebrates. From data mining, we found a novel prmt8e6+43 splicing variant with a 43-nucleotide (nt) extension at the 5' of exon 6 in chicken. RT-PCR analyses confirmed the existence of two splicing variants but also detected a third upper signal. The triplet pattern detected in chicken suggests that one strand from the prmt8e6+43 transcript and one strand from the regular splicing products form a heteroduplex with a bulb conformation and the two transcripts are of similar abundance. One short plus one faint upper heteroduplex signal detected in mouse and human indicate that the level of the variant is much less than the normal one in mammals. The relative expression of the normal and prmt8e6+43 variants in different species can be inferred from the reads of intron 5 that contains the 43-nt extension or not in the RNA-seq data of NCBI Gene database. The results of the analyses showed that the prmt8e6+43 variant is relatively abundant in birds but much less or even not detected in mammalian species. As conserved intron 5 sequences and evidences of alternative splicing (AS) are detected in elephant shark, a cartilaginous fish with the slowest-evolving genome, we propose that the prmt8e6+43 variant is present in the common ancestor of jawed vertebrates. The prmt8e6+43 variant includes a premature termination codon and thus should encode a truncated PRMT8 with deletion from the dimerization arm. Western blot analyses showed very weak low-molecular-weight signals in chicken, which might be the C-terminal truncated PRMT8. Why avian species maintain high RNA but not protein levels of the prmt8e6+43 variant and whether the evolutionary conserved sequence and AS might regulate PRMT8 expression require further investigation.


Assuntos
Processamento Alternativo/genética , Aves/genética , Variação Genética , Íntrons/genética , Proteína-Arginina N-Metiltransferases/genética , Sítios de Splice de RNA/genética , Vertebrados/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas/genética , Humanos , Camundongos , Nucleotídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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