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1.
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
2.
Mol Biol (Mosk) ; 53(3): 524-528, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184618

RESUMO

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle orN-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.


Assuntos
Proteínas/análise , Proteínas/química , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Sequências Repetitivas de Aminoácidos/genética , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas/genética , Proteoma/análise , Proteoma/química , Proteoma/genética , Processamento de RNA/genética
3.
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
4.
PLoS Genet ; 15(5): e1008130, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048900

RESUMO

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.


Assuntos
Glaucoma de Ângulo Fechado/genética , Hiperopia/genética , Proteínas de Membrana/genética , Microftalmia/genética , Degeneração Retiniana/genética , Fatores de Transcrição/genética , Adulto , Animais , Criança , Pré-Escolar , Éxons , Família , Feminino , Mutação da Fase de Leitura/genética , Variação Genética/genética , Glaucoma de Ângulo Fechado/metabolismo , Humanos , Hiperopia/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/metabolismo , Pessoa de Meia-Idade , Linhagem , Sítios de Splice de RNA/genética , Erros de Refração/genética , Fatores de Transcrição/metabolismo
5.
Nat Commun ; 10(1): 1777, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992453

RESUMO

Nerve conduction (NC) studies generate measures of peripheral nerve function that can reveal underlying pathology due to axonal loss, demyelination or both. We perform a genome-wide association study of sural NC amplitude and velocity in 7045 Icelanders and find a low-frequency splice-donor variant in PRPH (c.996+1G>A; MAF = 1.32%) associating with decreased NC amplitude but not velocity. PRPH encodes peripherin, an intermediate filament (IF) protein involved in cytoskeletal development and maintenance of neurons. Through RNA and protein studies, we show that the variant leads to loss-of-function (LoF), as when over-expressed in a cell line devoid of other IFs, it does not allow formation of the normal filamentous structure of peripherin, yielding instead punctate protein inclusions. Recall of carriers for neurological assessment confirms that from an early age, homozygotes have significantly lower sural NC amplitude than non-carriers and are at risk of a mild, early-onset, sensory-negative, axonal polyneuropathy.


Assuntos
Condução Nervosa/genética , Periferinas/genética , Polineuropatias/genética , Sítios de Splice de RNA/genética , Nervo Sural/fisiopatologia , Adulto , Idade de Início , Idoso , Axônios/patologia , Estudos de Casos e Controles , Linhagem Celular , Feminino , Seguimentos , Estudo de Associação Genômica Ampla , Homozigoto , Humanos , Islândia/epidemiologia , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Polineuropatias/epidemiologia , Polineuropatias/fisiopatologia , Prevalência , Processamento de RNA/fisiologia
6.
Genome Res ; 29(5): 711-722, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962178

RESUMO

The inclusion of exons during the splicing process depends on the binding of splicing factors to short low-complexity regulatory sequences. The relationship between exonic splicing regulatory sequences and coding sequences is still poorly understood. We demonstrate that exons that are coregulated by any given splicing factor share a similar nucleotide composition bias and preferentially code for amino acids with similar physicochemical properties because of the nonrandomness of the genetic code. Indeed, amino acids sharing similar physicochemical properties correspond to codons that have the same nucleotide composition bias. In particular, we uncover that the TRA2A and TRA2B splicing factors that bind to adenine-rich motifs promote the inclusion of adenine-rich exons coding preferentially for hydrophilic amino acids that correspond to adenine-rich codons. SRSF2 that binds guanine/cytosine-rich motifs promotes the inclusion of GC-rich exons coding preferentially for small amino acids, whereas SRSF3 that binds cytosine-rich motifs promotes the inclusion of exons coding preferentially for uncharged amino acids, like serine and threonine that can be phosphorylated. Finally, coregulated exons encoding amino acids with similar physicochemical properties correspond to specific protein features. In conclusion, the regulation of an exon by a splicing factor that relies on the affinity of this factor for specific nucleotide(s) is tightly interconnected with the exon-encoded physicochemical properties. We therefore uncover an unanticipated bidirectional interplay between the splicing regulatory process and its biological functional outcome.


Assuntos
Processamento Alternativo , Éxons/genética , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA/metabolismo , Aminoácidos/química , Composição de Bases/genética , Linhagem Celular , Código Genético , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Íntrons/genética , Motivos de Nucleotídeos/genética , Análise de Sequência de Proteína , Análise de Sequência de RNA , Fatores de Processamento de Serina-Arginina/metabolismo
7.
Nucleic Acids Res ; 47(10): e59, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30869147

RESUMO

Deletions in the 16.6 kb mitochondrial genome have been implicated in numerous disorders that often display muscular and/or neurological symptoms due to the high-energy demands of these tissues. We describe a catalogue of 4489 putative mitochondrial DNA (mtDNA) deletions, including their frequency and relative read rate, using a combinatorial approach of mitochondria-targeted PCR, next-generation sequencing, bioinformatics, post-hoc filtering, annotation, and validation steps. Our bioinformatics pipeline uses MapSplice, an RNA-seq splice junction detection algorithm, to detect and quantify mtDNA deletion breakpoints rather than mRNA splices. Analyses of 93 samples from postmortem brain and blood found (i) the 4977 bp 'common deletion' was neither the most frequent deletion nor the most abundant; (ii) brain contained significantly more deletions than blood; (iii) many high frequency deletions were previously reported in MitoBreak, suggesting they are present at low levels in metabolically active tissues and are not exclusive to individuals with diagnosed mitochondrial pathologies; (iv) many individual deletions (and cumulative metrics) had significant and positive correlations with age and (v) the highest deletion burdens were observed in major depressive disorder brain, at levels greater than Kearns-Sayre Syndrome muscle. Collectively, these data suggest the Splice-Break pipeline can detect and quantify mtDNA deletions at a high level of resolution.


Assuntos
Biologia Computacional/métodos , DNA Mitocondrial/genética , Transtorno Depressivo Maior/genética , Sítios de Splice de RNA/genética , Análise de Sequência de RNA/métodos , Deleção de Sequência , Algoritmos , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Quebras de DNA , DNA Mitocondrial/química , Transtorno Depressivo Maior/sangue , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase
8.
Mol Immunol ; 107: 91-96, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30685616

RESUMO

Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3' splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3' ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3' ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3' ss seems to be co-regulated with the authentic 3' ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.


Assuntos
Processamento Alternativo/genética , Proteína Inibidora do Complemento C1/genética , Éxons/genética , Sítios de Splice de RNA/genética , Sequência de Bases , Núcleo Celular/genética , Células Hep G2 , Humanos , Mutação/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Interferente Pequeno/metabolismo
9.
Int J Oncol ; 54(3): 859-868, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664192

RESUMO

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three­generation family with HME. Bioinformatics and TA clone­sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) revealed that the expression levels of wild­type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1­DEL) and full­length EXT1 mRNA (EXT1­FL) were overexpressed in 293­T cells and Cos­7 cells using lentivirus infection. RT­qPCR demonstrated that the expression level of EXT1­DEL was significantly increased, compared with EXT1­FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1­DEL was detected by western blot analysis, and the level was increased, compared with EXT1­FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1­DEL was located in the cytoplasm of Cos­7 cells, which was consistent with the localization of the EXT1­FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1­DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.


Assuntos
Exostose Múltipla Hereditária/genética , N-Acetilglucosaminiltransferases/genética , Processamento de RNA/genética , Adulto , Idoso , Linhagem Celular Transformada , Citoplasma/metabolismo , Exostose Múltipla Hereditária/metabolismo , Exostose Múltipla Hereditária/patologia , Feminino , Expressão Gênica , Estudos de Associação Genética , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , N-Acetilglucosaminiltransferases/metabolismo , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismo
10.
J Hum Genet ; 64(4): 341-346, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30692597

RESUMO

The genetic causes of combined pituitary hormone deficiency remain elusive in most patients. Recently, incompletely penetrant heterozygous mutations in ROBO1 have been described in patients with pituitary stalk interruption syndrome. Herein, we identified a novel homozygous slice site mutation in ROBO1 (c.1342+1G>A) using a trio whole-exome sequencing strategy in a 5-year-old Japanese boy who had combined pituitary hormone deficiency, psychomotor developmental delay, severe intellectual disability, sensorineural hearing loss, strabismus, and characteristic facial features, including a broad forehead, micrognathia, and arched eyebrows. Magnetic resonance imaging delineated anterior pituitary hypoplasia, ectopic posterior pituitary, invisible pituitary stalk, thinning of the corpus callosum, and hypoplasia of the pons and midbrain. The phenotypically normal parents (first cousins) were heterozygous for the mutation. The results provide further evidence of ROBO1 being involved in the development of the pituitary gland. A recessive mutation of ROBO1 is a potential novel cause of a syndromic disorder associated with combined pituitary hormone deficiency.


Assuntos
Perda Auditiva Neurossensorial/genética , Hipopituitarismo/genética , Deficiência Intelectual/genética , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Pré-Escolar , Corpo Caloso/diagnóstico por imagem , Corpo Caloso/fisiopatologia , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/diagnóstico por imagem , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Hipopituitarismo/complicações , Hipopituitarismo/diagnóstico por imagem , Hipopituitarismo/fisiopatologia , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/fisiopatologia , Imagem por Ressonância Magnética , Masculino , Mutação , Sítios de Splice de RNA/genética , Sequenciamento Completo do Exoma
11.
Clin Chim Acta ; 491: 15-18, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30639237

RESUMO

Primary autosomal recessive microcephaly (MCPH) is a rare hereditary disease characterized by congenitally small with brain circumference of the head below 3 standard deviations (SD). By far, 18 MCPH genes have been reported to be associated with the disease. SASS6 gene functioned in assembly of centrioles that the majority of MCPH genes present at the centrosome. There was only research reporting a homozygous missense mutation in SASS6 gene detected in a consanguineous Pakistani family. By conducting Whole-exome sequencing (WES) and Sanger sequencing on the family trio, we identified two novel splice site mutations c.127-13A>G and c.1867+2T>A in compound heterozygous hereditary form in the SASS6 gene. The two mutations were confirmed to alter mRNA splicing by RT-PCR assay. Our finding supported the role of SASS6 in the pathogenesis of microcephaly, expanding mutation spectrums and contributing to understanding of molecular mechanisms of MCPH.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Proteínas de Ciclo Celular/genética , Heterozigoto , Microcefalia/genética , Mutação , Linhagem , Adulto , Sequência de Bases , Feminino , Humanos , Masculino , Sítios de Splice de RNA/genética
12.
Mol Genet Genomics ; 294(2): 493-500, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30604070

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUT) are a wide range of congenital structural renal defects. CAKUT is the leading cause of chronic renal failure and end-stage renal disease in children. Studies in humans and animal models have confirmed the large genetic contribution to CAKUT. The previous evidence suggested that human TBX6 coding mutations might cause CAKUT via gene-dosage insufficiency. However, the potential involvement of TBX6 noncoding mutations in CAKUT remains to be elucidated. Here, we described DNA sequencing and copy-number analysis of TBX6 in 269 Chinese subjects with CAKUT. Interestingly, we identified two heterozygous noncoding variants of TBX6 in sporadic subjects with CAKUT: one is c.769-7delT, from a subject with duplex renal and collecting system, and the other is a 3' untranslated region (3'-UTR) variant (c.1392C>T) from a subject with unilateral renal hypoplasia. These two TBX6 noncoding variants are novel and extremely rare, respectively, in human populations archived in the ExAC database. The mini-gene splicing assay showed that the TBX6 c.769-7delT variant significantly reduced the splicing efficiency of TBX6 intron 5 when compared to the wild-type control. In this work, we identified a novel splicing variant of TBX6 in human CAKUT. Our experimental observations suggested that the TBX6 noncoding variant can affect gene expression and may potentially be involved in human CAKUT.


Assuntos
Sítios de Splice de RNA/genética , Análise de Sequência de DNA , Proteínas com Domínio T/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Adolescente , Criança , Feminino , Humanos , Rim/fisiopatologia , Masculino , Mutação , Fenótipo , Anormalidades Urogenitais/fisiopatologia , Refluxo Vesicoureteral/fisiopatologia , Sequenciamento Completo do Exoma
13.
Mol Genet Genomic Med ; 7(3): e552, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30688039

RESUMO

BACKGROUND: Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next-generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain. METHODS: We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT-PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency. RESULTS: The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT-PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca++ ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts. CONCLUSIONS: This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia.


Assuntos
Biologia Computacional/métodos , Testes Genéticos/métodos , Miotonia Congênita/genética , Sítios de Splice de RNA/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Sequenciamento Completo do Exoma/métodos , Algoritmos , Animais , Células Cultivadas , Drosophila melanogaster , Humanos , Masculino , Músculo Esquelético/metabolismo , Mutação , Miotonia Congênita/patologia , Fenótipo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adulto Jovem
14.
Nature ; 565(7741): 606-611, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651636

RESUMO

Spliceosomal introns are ubiquitous non-coding RNAs that are typically destined for rapid debranching and degradation. Here we describe 34 excised introns in Saccharomyces cerevisiae that-despite being rapidly degraded in log-phase growth-accumulate as linear RNAs under either saturated-growth conditions or other stresses that cause prolonged inhibition of TORC1, which is a key integrator of growth signalling. Introns that become stabilized remain associated with components of the spliceosome and differ from other spliceosomal introns in having a short distance between their lariat branch point and 3' splice site, which is necessary and sufficient for their stabilization. Deletion of these unusual introns is disadvantageous in saturated conditions and causes aberrantly high growth rates in yeast that are chronically challenged with the TORC1 inhibitor rapamycin. The reintroduction of native or engineered stable introns suppresses this aberrant rapamycin response. Thus, excised introns function within the TOR growth-signalling network of S. cerevisiae and, more generally, excised spliceosomal introns can have biological functions.


Assuntos
Íntrons/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Actinas/genética , Genes Fúngicos/genética , Aptidão Genética , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Sítios de Splice de RNA/genética , Estabilidade de RNA , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Spliceossomos/metabolismo
15.
Invest Ophthalmol Vis Sci ; 60(1): 282-293, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30657523

RESUMO

Purpose: To gain insight into the pathophysiology of vitreoretinal degeneration, the clinical course of three family members with Versican Vitreoretinopathy (VVR) is described, and a canonical splice site mutation in the gene encoding for versican (VCAN) protein was biochemically analyzed. Methods: A retrospective chart review, human eye histopathology, Sanger DNA sequencing, protein structural modeling, and in vitro proteolysis assays were performed. Results: The proband (II:1), mother (I:2), and younger sibling (II:2) suffered retinal degeneration with foveal sparing and retinal detachments with proliferative vitreoretinopathy, features that were confirmed on histopathologic analysis. All affected members carried a heterozygous adenine to guanine variant (c.4004-2A>G) predicted to result in exon 8 skipping or the deletion of 13 amino acids at the beginning of the GAGß chain (VCAN p.1335-1347). This deleted region corresponded to a putative MMP cleavage site, validated using fluorescence resonance energy transfer (FRET)-based proteolysis assays. Proteomic network analysis identified 10 interacting partners in the human vitreous and retina linked to retinal detachment and degeneration. Conclusions: VVR causes significant ocular disease, including retinal detachment and retinal dystrophy. The intronic VCAN mutation removes an MMP cleavage site, which alters versican structure and results in abnormal vitreous modeling. Disruption of a versican protein network may underlie clinicopathologic disease features and point to targeted therapies.


Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Mutação/genética , Sítios de Splice de RNA/genética , Degeneração Retiniana/genética , Versicanas/deficiência , Adulto , Idoso , Eletrorretinografia , Feminino , Humanos , Masculino , Linhagem , Proteólise , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Estudos Retrospectivos , Versicanas/genética , Versicanas/metabolismo , Acuidade Visual , Corpo Vítreo/patologia
16.
Cell ; 176(3): 535-548.e24, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30661751

RESUMO

The splicing of pre-mRNAs into mature transcripts is remarkable for its precision, but the mechanisms by which the cellular machinery achieves such specificity are incompletely understood. Here, we describe a deep neural network that accurately predicts splice junctions from an arbitrary pre-mRNA transcript sequence, enabling precise prediction of noncoding genetic variants that cause cryptic splicing. Synonymous and intronic mutations with predicted splice-altering consequence validate at a high rate on RNA-seq and are strongly deleterious in the human population. De novo mutations with predicted splice-altering consequence are significantly enriched in patients with autism and intellectual disability compared to healthy controls and validate against RNA-seq in 21 out of 28 of these patients. We estimate that 9%-11% of pathogenic mutations in patients with rare genetic disorders are caused by this previously underappreciated class of disease variation.


Assuntos
Previsões/métodos , Precursores de RNA/genética , Processamento de RNA/genética , Algoritmos , Processamento Alternativo/genética , Transtorno Autístico/genética , Aprendizado Profundo , Éxons/genética , Humanos , Deficiência Intelectual/genética , Íntrons/genética , Redes Neurais (Computação) , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Sítios de Splice de RNA/fisiologia
17.
Cell ; 176(3): 549-563.e23, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30661752

RESUMO

Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modeling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighboring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations.


Assuntos
Processamento Alternativo/genética , Processamento de RNA/genética , Receptor fas/genética , Animais , Éxons/genética , Técnicas Genéticas , Genética , Genótipo , Humanos , Íntrons/genética , Camundongos , Modelos Teóricos , Mutação/genética , Fenótipo , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismo
18.
Clin Dysmorphol ; 28(1): 22-25, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30407211

RESUMO

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is an autosomal recessive disorder characterized by gastrointestinal dysmotility, cachexia, ptosis, peripheral neuropathy and leukoencephalopathy. The diagnosis is often not made until 5-10 years after the onset of symptoms. MNGIE is caused by mutations in thymidine phosphorylase gene TYMP. Here, we present a 19-year-old boy with MNGIE who had a chronic intestinal pseudo-obstruction, and we describe his family history. Genetic analysis revealed a novel homozygous c.765+1G>C intronic mutation which is expected to disrupt splicing of TYMP in the patient. Family screening revealed that the brother was also affected and the mother was a carrier. MNGIE should be considered and genetic testing instigated if individuals with cachexia have neuromuscular complaints or symptoms of chronic intestinal pseudo-obstruction.


Assuntos
Pseudo-Obstrução Intestinal/complicações , Pseudo-Obstrução Intestinal/genética , Encefalomiopatias Mitocondriais/complicações , Encefalomiopatias Mitocondriais/genética , Mutação/genética , Sítios de Splice de RNA/genética , Sequência de Bases , Feminino , Humanos , Pseudo-Obstrução Intestinal/diagnóstico por imagem , Imagem por Ressonância Magnética , Masculino , Encefalomiopatias Mitocondriais/diagnóstico por imagem , Linhagem , Tomografia Computadorizada por Raios X , Adulto Jovem
19.
Methods Mol Biol ; 1881: 83-99, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30350199

RESUMO

Mutational landscape of CLL is now known to include recurrent non-synonymous mutations in SF3B1, a core splicing factor. About 5-10% of newly diagnosed CLL harbor these mutations which are typically limited to HEAT domains in the carboxyl-terminus of the protein. Importantly, the mutations are not specific to CLL but also present in several unrelated clonal disorders. Analysis of patient samples and cell lines has shown the primary splicing aberration in SF3B1-mutant cells to the use of novel or "cryptic" 3' splice sites (3SS). Advances in genome-editing and next-generation sequencing (NGS) have allowed development of isogenic models and detailed analysis of changes to the transcriptome with relative ease. In this manuscript, we focus on two relevant methods to study splicing factor mutations in CLL: development of isogenic scalable cell lines and informatics analysis of RNA-Seq datasets.


Assuntos
Biologia Computacional/métodos , Leucemia Linfocítica Crônica de Células B/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Análise de Sequência de RNA/métodos , Animais , Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular , Biologia Computacional/instrumentação , Conjuntos de Dados como Assunto , Edição de Genes/instrumentação , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Células-Tronco Embrionárias Murinas , Mutação , Domínios Proteicos/genética , Sítios de Splice de RNA/genética , Processamento de RNA/genética , Análise de Sequência de RNA/instrumentação , Software
20.
BMC Genomics ; 19(1): 980, 2018 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-30594132

RESUMO

BACKGROUND: Most eukaryotic genes comprise exons and introns thus requiring the precise removal of introns from pre-mRNAs to enable protein biosynthesis. U2 and U12 spliceosomes catalyze this step by recognizing motifs on the transcript in order to remove the introns. A process which is dependent on precise definition of exon-intron borders by splice sites, which are consequently highly conserved across species. Only very few combinations of terminal dinucleotides are frequently observed at intron ends, dominated by the canonical GT-AG splice sites on the DNA level. RESULTS: Here we investigate the occurrence of diverse combinations of dinucleotides at predicted splice sites. Analyzing 121 plant genome sequences based on their annotation revealed strong splice site conservation across species, annotation errors, and true biological divergence from canonical splice sites. The frequency of non-canonical splice sites clearly correlates with their divergence from canonical ones indicating either an accumulation of probably neutral mutations, or evolution towards canonical splice sites. Strong conservation across multiple species and non-random accumulation of substitutions in splice sites indicate a functional relevance of non-canonical splice sites. The average composition of splice sites across all investigated species is 98.7% for GT-AG, 1.2% for GC-AG, 0.06% for AT-AC, and 0.09% for minor non-canonical splice sites. RNA-Seq data sets of 35 species were incorporated to validate non-canonical splice site predictions through gaps in sequencing reads alignments and to demonstrate the expression of affected genes. CONCLUSION: We conclude that bona fide non-canonical splice sites are present and appear to be functionally relevant in most plant genomes, although at low abundance.


Assuntos
Genoma de Planta , Íntrons/genética , Plantas/genética , Sítios de Splice de RNA/genética , Processamento de RNA/genética , Éxons/genética , Estudo de Associação Genômica Ampla , Genômica
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