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1.
Adv Exp Med Biol ; 1164: 119-139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576545

RESUMO

Alternative splicing, the process of removing introns and joining exons of pre-mRNA, is critical for growth, development, tissue homeostasis, and species diversity. Dysregulation of alternative splicing can initiate and drive disease. Aberrant alternative splicing has been shown to promote the "hallmarks of cancer" in both hematological and solid cancers. Of interest, recent work has focused on the role of alternative splicing in prostate cancer and prostate cancer health disparities. We will provide a review of prostate cancer health disparities involving the African American population, alternative RNA splicing, and alternative splicing in prostate cancer. Lastly, we will summarize our work on differential alternative splicing in prostate cancer disparities and its implications for disparate health outcomes and therapeutic targets.


Assuntos
Processamento Alternativo , Resistência a Medicamentos , Disparidades nos Níveis de Saúde , Neoplasias da Próstata , Afro-Americanos/estatística & dados numéricos , Processamento Alternativo/genética , Resistência a Medicamentos/genética , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/fisiopatologia
2.
Genes Dev ; 33(17-18): 1221-1235, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371437

RESUMO

TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71's function in regulating stem cell fates.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Proteínas de Ligação a RNA/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Humanos , Camundongos , Camundongos Knockout , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos/genética , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo
3.
BMC Bioinformatics ; 20(1): 434, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438847

RESUMO

BACKGROUND: The epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells. Elevated expression levels of EGFR are associated with prognosis and clinical outcomes of patients in a variety of tumor types. There are at least four splice variants of the mRNA encoding four protein isoforms of EGFR in humans, named I through IV. EGFR isoform I is the full-length protein, whereas isoforms II-IV are shorter protein isoforms. Nevertheless, all EGFR isoforms bind the epidermal growth factor (EGF). Although EGFR is an essential target of long-established and successful tumor therapeutics, the exact function and biomarker potential of alternative EGFR isoforms II-IV are unclear, motivating more in-depth analyses. Hence, we analyzed transcriptome data from glioblastoma cell line SF767 to predict target genes regulated by EGFR isoforms II-IV, but not by EGFR isoform I nor other receptors such as HER2, HER3, or HER4. RESULTS: We analyzed the differential expression of potential target genes in a glioblastoma cell line in two nested RNAi experimental conditions and one negative control, contrasting expression with EGF stimulation against expression without EGF stimulation. In one RNAi experiment, we selectively knocked down EGFR splice variant I, while in the other we knocked down all four EGFR splice variants, so the associated effects of EGFR II-IV knock-down can only be inferred indirectly. For this type of nested experimental design, we developed a two-step bioinformatics approach based on the Bayesian Information Criterion for predicting putative target genes of EGFR isoforms II-IV. Finally, we experimentally validated a set of six putative target genes, and we found that qPCR validations confirmed the predictions in all cases. CONCLUSIONS: By performing RNAi experiments for three poorly investigated EGFR isoforms, we were able to successfully predict 1140 putative target genes specifically regulated by EGFR isoforms II-IV using the developed Bayesian Gene Selection Criterion (BGSC) approach. This approach is easily utilizable for the analysis of data of other nested experimental designs, and we provide an implementation in R that is easily adaptable to similar data or experimental designs together with all raw datasets used in this study in the BGSC repository, https://github.com/GrosseLab/BGSC .


Assuntos
Processamento Alternativo/genética , Biologia Computacional/métodos , Receptores ErbB/genética , Glioblastoma/genética , Teorema de Bayes , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Probabilidade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
4.
Nat Neurosci ; 22(10): 1709-1717, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451803

RESUMO

Nervous system function relies on complex assemblies of distinct neuronal cell types that have unique anatomical and functional properties instructed by molecular programs. Alternative splicing is a key mechanism for the expansion of molecular repertoires, and protein splice isoforms shape neuronal cell surface recognition and function. However, the logic of how alternative splicing programs are arrayed across neuronal cells types is poorly understood. We systematically mapped ribosome-associated transcript isoforms in genetically defined neuron types of the mouse forebrain. Our dataset provides an extensive resource of transcript diversity across major neuron classes. We find that neuronal transcript isoform profiles reliably distinguish even closely related classes of pyramidal cells and inhibitory interneurons in the mouse hippocampus and neocortex. These highly specific alternative splicing programs selectively control synaptic proteins and intrinsic neuronal properties. Thus, transcript diversification via alternative splicing is a central mechanism for the functional specification of neuronal cell types and circuits.


Assuntos
Processamento Alternativo/genética , Neurônios/fisiologia , Ribossomos/genética , Transcrição Genética/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neurônios/classificação , Terminações Pré-Sinápticas/metabolismo , Prosencéfalo/citologia , Isoformas de Proteínas/genética , Células Piramidais/fisiologia
5.
Mol Biol (Mosk) ; 53(3): 411-420, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184606

RESUMO

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.


Assuntos
Processamento Alternativo/genética , Antitrombina III/genética , Sítios de Splice de RNA/genética , Éxons/genética , Humanos , Íntrons/genética
6.
PLoS Genet ; 15(6): e1008226, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199789

RESUMO

Carbonic anhydrase-8 (CA8) is an intracellular protein that functions as an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1) critical to intracellular Ca++ release, synaptic functions and neuronal excitability. We showed previously that murine nociception and analgesic responses are regulated by the expression of this gene in dorsal root ganglion (DRG) associated with a cis-eQTL. In this report, we identify an exon-level cis-eQTL (rs6471859) that regulates human DRG CA8 alternative splicing, producing a truncated 1,697bp transcript (e.g., CA8-204). Our functional genomic studies show the "G" allele at rs6471859 produces a cryptic 3'UTR splice site regulating expression of CA8-204. We developed constructs to study the expression and function of the naturally occurring CA8-204G transcript (G allele at rs6471859), CA8-204C (C allele at rs6471859 reversion mutation) and CA8-201 (full length transcript). CA8-204G transcript expression occurred predominantly in non-neuronal cells (HEK293), while CA8-204C expression was restricted to neuronal derived cells (NBL) in vitro. CA8-204G produced a stable truncated transcript in HEK293 cells that was barely detectable in NBL cells. We also show CA8-204 produces a stable peptide that inhibits pITPR1 and Ca++ release in HEK293 cells. These results imply homozygous G/G individuals at rs6471859, which are common in the general population, produce exclusively CA8-204G that is barely detectable in neuronal cells. CA8 null mutations that greatly impact neuronal functions are associated with severe forms of spinal cerebellar ataxia, and our data suggest G/G homozygotes should display a similar phenotype. To address this question, we show in vivo using AAV8-FLAG-CA8-204G and AAV8-V5-CA8-201 gene transfer delivered via intra-neural sciatic nerve injection (SN), that these viral constructs are able to transduce DRG cells and produce similar analgesic and anti-hyperalgesic responses to inflammatory pain. Immunohistochemistry (IHC) examinations of DRG tissues further show CA8-204G peptide is expressed in advillin expressing neuronal cells, but to a lesser extent compared to glial cells. These findings explain why G/G homozygotes that exclusively produce this truncated functional peptide in DRG evade a severe phenotype. These genomic studies significantly advance the literature regarding structure-function studies on CA8-ITPR1 critical to calcium signaling pathways, synaptic functioning, neuronal excitability and analgesic responses.


Assuntos
Biomarcadores Tumorais/genética , Sinalização do Cálcio/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Neurônios/metabolismo , Dor/genética , Processamento Alternativo/genética , Animais , Biomarcadores Tumorais/farmacologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Camundongos , Mutação/genética , Neurônios/patologia , Especificidade de Órgãos , Dor/patologia , Peptídeos/genética , Peptídeos/farmacologia , Locos de Características Quantitativas/genética , Sítios de Splice de RNA/genética , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo
7.
RNA ; 25(9): 1202-1210, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31151991

RESUMO

Reverse transcription of RNA is fallible, introducing biases and confounding the quantification of transcript abundance. We demonstrate that circular RNAs (circRNAs) are more subjective to overestimation of transcript abundance than cognate linear RNAs due to their covalently closed, circular form, producing multiple concatameric products from a single priming of reverse transcriptase. We developed SplintQuant, where custom DNA oligonucleotides are ligated by PBCV-1 DNA ligase only when bound to their target RNA. These circRNA-specific DNA oligonucleotides are terminally tagged with universal primers, allowing SplintQuant to accurately quantify even lowly abundant circRNAs through highly specific quantitative PCR (qPCR) in the absence of reverse transcription. SplintQuant is sensitive, specific, highly reproducible, and applicable to the quantification of canonical and noncanonical RNA transcripts including alternative splice variants, gene fusions, and offers a gold-standard approach for accurately quantifying circRNAs.


Assuntos
RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/genética , Processamento Alternativo/genética , Viés , Linhagem Celular , Humanos , Análise de Sequência de RNA/métodos
8.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
9.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086020

RESUMO

Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.


Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Processamento de RNA/genética , Spodoptera/genética , Processamento Alternativo/genética , Animais , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Ensaio de Desvio de Mobilidade Eletroforética , Éxons/genética , Feminino , Masculino , Ligação Proteica
10.
BMC Bioinformatics ; 20(1): 231, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068132

RESUMO

BACKGROUND: In eukaryotes, most genes code for multiple transcript isoforms that are generated through the complex and tightly regulated process of RNA splicing. Despite arising from identical precursor transcripts, alternatively spliced RNAs can have dramatically different functions. Transcriptome complexity is elevated further by the production of circular RNAs (circRNAs), another class of mature RNA that results from the splicing of a downstream splice donor to an upstream splice acceptor. While there has been a rapid expansion of circRNA catalogs in the last few years through the utilization of next generation sequencing approaches, our understanding of the mechanisms and regulation of circular RNA biogenesis, the impact that circRNA generation has on parental transcript processing, and the functions carried out by circular RNAs remains limited. RESULTS: Here, we present a visualization and analysis tool, SpliceV, that rapidly plots all relevant forward- and back-splice data, with exon and single nucleotide level coverage information from RNA-seq experiments in a publication quality format. SpliceV also integrates analysis features that assist investigations into splicing regulation and transcript functions through the display of predicted RNA binding protein sites and the configuration of repetitive elements along the primary transcript. CONCLUSIONS: SpliceV is an easy-to-use splicing visualization tool, compatible with both Python 2.7 and 3+, and distributed under the GNU Public License. The source code is freely available for download at https://github.com/flemingtonlab/SpliceV and can be installed from PyPI using pip.


Assuntos
Processamento Alternativo/genética , Éxons/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA/genética
11.
Gene ; 706: 140-145, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31078657

RESUMO

BACKGROUND: Alternative splicing regulates most of protein-coding genes by producing diverse messenger RNA transcripts; and mis-splicing events can induce aberrant protein isoforms that contribute to cancer development. It is possible that genetic variations in splicing associated genes may regulate the formation of transcripts and multiple protein isoforms by affecting the splice regulatory elements. In this study, we aimed to determine whether genetic variations in the crucial alternative-splicing genes were associated with breast cancer risk. MATERIALS AND METHODS: A case-control study was conducted with 1064 breast cancer cases and 1073 healthy controls from China. A total of 16 tagging polymorphisms within three splicing factor-associated genes (SFRS3, ESRP1 and ESRP2) were genotyped by using Infinium BeadChip. The association between the polymorphisms and risk of breast cancer was evaluated by computing odds ratios (OR) and 95% confidence intervals (CIs). RESULTS: The genotype distribution of rs2145048 in SFRS3 was different between cases and controls (Bonferroni corrected P = 0.022). After adjusting for age, age at menarche and menopausal status, the A allele of rs2145048 showed an inverse association with breast cancer risk in the additive model (adjusted OR = 0.81, 95% CI = 0.71-0.92, P = 0.001, Bonferroni corrected P = 0.016). In the stratification analysis, the association between rs2145048 A allele and breast cancer remained significant in subgroups of earlier menarche, older first born, premenopausal status, and ER/PR negative status. CONCLUSIONS: This study provided the first evidence that SFRS3 rs2145048 was associated with breast cancer susceptibility in Chinese women, which might represent a biomarker to improve the identification of individuals at high risk of this malignancy.


Assuntos
Neoplasias da Mama/genética , Fatores de Processamento de Serina-Arginina/genética , Adulto , Alelos , Processamento Alternativo/genética , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Genótipo , Humanos , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Fatores de Risco , Fatores de Processamento de Serina-Arginina/fisiologia
12.
Gene ; 709: 17-24, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31102716

RESUMO

Angiopoietin-like protein 6, which is encoded by ANGPTL6 gene (also known as angiopoietin growth factor, AGF), has been extensively characterized with regard to its proposed functions as angiogenesis and energy metabolism. The present results showed the occurrence of alternative splicing by intron retention (IR) event in the bovine ANGPTL6 gene (bANGPTL6). By means of RT-PCR, TA clone and sequencing, we have shown that the bANGPTL6 gene has a splice variant generated by the retention of its partial intron 3. The computational analysis of the bANGPTL6 genomic sequence showed that its intron 3 has a high percentage of GC (62.31%) and a length of 199 nt, characteristics that have been associated with an IR event. The IR event does not interfere with the coding region as the bANGPTL6 prepropeptide is entirely coded in the third exon. Additionally, both the intronless (namely, bANGPTL6α) and intron-retaining (namely, bANGPTL6ß) ANGPTL6 transcripts are constitutively co-expressed in the bovine liver. Further, the relative expression level of different variants in liver was tested by both semi-RT-PCR and RT-qPCR methods. The results suggested bANGPTL6ß are significantly higher than bANGPTL6α. Overall, our findings will be helpful for studies on the molecular mechanism of IR events and the functions of ANGPTL6 gene. Specially, bANGPTL6ß gene probably contributes to a new target for treatment of obesity and obesity-related diseases.


Assuntos
Processamento Alternativo/genética , Proteínas Semelhantes a Angiopoietina/genética , Bovinos/genética , Íntrons/genética , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Expressão Gênica , Fígado/metabolismo , Isoformas de Proteínas/genética
13.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027366

RESUMO

Alternative splicing of pre-mRNA allows the generation of multiple splice isoforms from a given gene, which can have distinct functions. In fact, splice isoforms can have opposing functions and there are many instances whereby a splice isoform acts as an inhibitor of canonical isoform function, thereby adding an additional layer of regulation to important processes. Angiogenesis is an important process that is governed by alternative splicing mechanisms. This review focuses on the alternative spliced isoforms of key genes that are involved in the angiogenesis process; VEGF-A, VEGFR1, VEGFR2, NRP-1, FGFRs, Vasohibin-1, Vasohibin-2, HIF-1α, Angiopoietin-1 and Angiopoietin-2.


Assuntos
Processamento Alternativo/fisiologia , Neovascularização Patológica/metabolismo , Processamento Alternativo/genética , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Humanos , Neovascularização Patológica/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo
14.
Int J Mol Sci ; 20(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010208

RESUMO

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by "back-splicing", which is the linking covalently of 3'- and 5'-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.


Assuntos
Regulação da Expressão Gênica , Distrofia Miotônica/genética , RNA/genética , Adulto , Processamento Alternativo/genética , Estudos de Casos e Controles , Linhagem Celular , Feminino , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Miotônica/sangue , Reação em Cadeia da Polimerase , RNA/sangue , Reprodutibilidade dos Testes
15.
Nat Cell Biol ; 21(5): 640-650, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011167

RESUMO

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Fator de Processamento U2AF/genética , Processamento Alternativo/genética , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata/genética , Inflamação/genética , Inflamação/patologia , Leucemia Mieloide Aguda/patologia , Masculino , Mutação/genética , Síndromes Mielodisplásicas/patologia , Isoformas de Proteínas/genética , Transdução de Sinais , Spliceossomos/genética
16.
Pestic Biochem Physiol ; 156: 56-62, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027581

RESUMO

Glutamate-gated chloride channels (GluCls) mediate inhibitory synaptic transmission in invertebrate nervous systems, and only one GluCl gene has been found in insects. Therefore, insect GluCls are one of the major targets of insecticides including avermectins. In the present study, a 1347 bp full-length cDNA encoding a 449-amino acid protein (named MsGluCl, GenBank ID: MK336885) was cloned from the oriental armyworm, Mythimna separata, and characterized two alternative splicing variants of MsGluCl. The protein shares 76.9-98.6% identity with other insect GluCl isoforms. Spatial and temporal expression analysis revealed that MsGluCl was highly expressed in the 3rd instar and adult head. Dietary ingestion of dsMsGluCl significantly reduced the mRNA level of MsGluCl and decreased abamectin mortality. Thus, our results reveal that MsGluCl could be the molecular target of abamectin and provide the basis for further understanding the resistance mechanism to abamectin in arthropods.


Assuntos
Processamento Alternativo/genética , Canais de Cloreto/metabolismo , Clonagem Molecular/métodos , Mariposas/genética , Animais , Canais de Cloreto/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Inseticidas/farmacologia , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Mariposas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Pediatr Cardiol ; 40(5): 1084-1091, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955100

RESUMO

Congenital heart defects (CHDs) are the leading cause of death in infants under 1 year of age. Aberrations in the expression and function of cardiac transcription factors (TFs) are a major contributor to CHDs. Despite the numerous studies undertaken to functionally characterize these TFs, their exact role in different stages of cardiogenesis is still not fully elucidated. Here we focused on HEY2, a basic helix loop helix transcriptional repressor, and its potential role in human ventricular septal defects. Genetic analysis was performed based on sequencing of DNA and cDNA obtained from post-operational cardiac tissues and blood of 17 Lebanese patients with various CHDs. The screen covered the entire coding regions of the GATA4, NKX2.5, TBX5, TBX20 and HEY2 genes. Our results revealed two novel somatic mutations, namely p.Ala229Thr and p.161_190 del, affecting HEY2 in the diseased cardiac tissues of two patients with VSD. These results suggest a potential role of HEY2 in regulating ventricular septation in humans.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Comunicação Interventricular/genética , Mutação/genética , Isoformas de Proteínas/genética , Proteínas Repressoras , Processamento Alternativo/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Análise Mutacional de DNA , Humanos , Lactente , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
18.
Gene ; 699: 135-144, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30849541

RESUMO

Next-generation sequencing (NGS) technology is highly expected to help researchers disclose the complexity of alternative splicing and understand its association with carcinogenesis. Alternative splicing alterations are firmly associated with multiple malignancies, in terms of functional roles in malignant transformation, motility, and/or metastasis of cancer cells. One perfect example illustrating the connection between alternative splicing and cancer is the human protein arginine methyltransferase 1 (PRMT1) gene, previously cloned from members of our research group and involved in a variety of processes including transcription, DNA repair, and signal transduction. Two splice variants of PRMT1 (variants v.1 and v.2) are downregulated in breast cancer. In addition, PRMT1 v.2 promotes the survival and invasiveness of breast cancer cells, while it could serve as a biomarker of unfavorable prognosis in colon cancer patients. The aim of this study was the molecular cloning of novel alternative splice variants of PRMT1 with the use of 3' RACE coupled with NGS technology. Extensive bioinformatics and computational analysis revealed a significant number of 19 novel alternative splicing events between annotated exons of PRMT1 as well as one novel exon, resulting in the discovery of multiple PRMT1 transcripts. In order to validate the full sequence of the novel transcripts, RT-PCR was carried out with the use of variant-specific primers. As a result, 58 novel PRMT1 transcripts were identified, 34 of which are mRNAs encoding new protein isoforms, whereas the rest 24 transcripts are candidates for nonsense-mediated mRNA decay (NMD).


Assuntos
Processamento Alternativo/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Linhagem Celular , Linhagem Celular Tumoral , Clonagem Molecular/métodos , Biologia Computacional/métodos , Éxons/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Isoformas de Proteínas/genética , RNA Mensageiro/genética
19.
Pestic Biochem Physiol ; 155: 72-80, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30857629

RESUMO

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems, and are of considerable interest in insecticide discovery. The full length cDNA encoding CsGluCl was cloned from the rice stem borer Chilo suppressalis (Walker). Multiple cDNA sequence alignment revealed three variants of CsGluCl generated by alternative splicing of exon 3 and exon 9. While all the transcripts were predominantly expressed in both nerve cord and brain, the expression patterns of these three variants differed among other tissues and developmental stages. Specifically, the expression level of CsGluCl C in cuticle was similar to that in nerve cord and brain, and was the predominant variant in late pupae and early adult stages. Both injection and oral delivery of dsGluCl significantly reduced the mRNA level of CsGluCl. Increased susceptibility to abamectin and reduced larvae growth and pupation rate were observed in dsGluCl-treated larvae. Thus, our results provide the evidence that in addition to act as the target of abamectin, GluCls also play important physiological roles in the development of insects.


Assuntos
Processamento Alternativo/genética , Canais de Cloreto/metabolismo , Ivermectina/análogos & derivados , Animais , Canais de Cloreto/genética , Éxons/genética , Ivermectina/farmacologia , Larva , Interferência de RNA , RNA Mensageiro/genética
20.
BMC Bioinformatics ; 20(Suppl 3): 133, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925859

RESUMO

BACKGROUND: The inference of splicing orthology relationships between gene transcripts is a basic step for the prediction of transcripts and the annotation of gene structures in genomes. The splicing structure of a sequence refers to the exon extremity information in a CDS or the exon-intron extremity information in a gene sequence. Splicing orthologous CDS are pairs of CDS with similar sequences and conserved splicing structures from orthologous genes. Spliced alignment that consists in aligning a spliced cDNA sequence against an unspliced genomic sequence, constitutes a promising, yet unexplored approach for the identification of splicing orthology relationships. Existing spliced alignment algorithms do not exploit the information on the splicing structure of the input sequences, namely the exon structure of the cDNA sequence and the exon-intron structure of the genomic sequences. Yet, this information is often available for coding DNA sequences (CDS) and gene sequences annotated in databases, and it can help improve the accuracy of the computed spliced alignments. To address this issue, we introduce a new spliced alignment problem and a method called SplicedFamAlign (SFA) for computing the alignment of a spliced CDS against a gene sequence while accounting for the splicing structures of the input sequences, and then the inference of transcript splicing orthology groups in a gene family based on spliced alignments. RESULTS: The experimental results show that SFA outperforms existing spliced alignment methods in terms of accuracy and execution time for CDS-to-gene alignment. We also show that the performance of SFA remains high for various levels of sequence similarity between input sequences, thanks to accounting for the splicing structure of the input sequences. It is important to notice that unlike all current spliced alignment methods that are meant for cDNA-to-genome alignments and can be used for CDS-to-gene alignments, SFA is the first method specifically designed for CDS-to-gene alignments. CONCLUSION: We show the usefulness of SFA for the comparison of genes and transcripts within a gene family for the purpose of analyzing splicing orthologies. It can also be used for gene structure annotation and alternative splicing analyses. SplicedFamAlign was implemented in Python. Source code is freely available at https://github.com/UdeS-CoBIUS/SpliceFamAlign .


Assuntos
Algoritmos , Processamento Alternativo/genética , Fases de Leitura Aberta/genética , Alinhamento de Sequência/métodos , Sequência de Bases , Simulação por Computador , Éxons/genética , Íntrons/genética , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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