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1.
Nucleic Acids Res ; 48(18): 10368-10382, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32955563

RESUMO

Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed.


Assuntos
Sítios Internos de Entrada Ribossomal/genética , Metiltransferases/genética , Biossíntese de Proteínas , RNA Circular/genética , Sequência de Bases/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , MicroRNAs/genética , Sítios de Splice de RNA/genética , RNA Circular/classificação , Proteínas de Ligação a RNA/genética
2.
Nat Commun ; 11(1): 4744, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958768

RESUMO

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.


Assuntos
Sítios de Splice de RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Cristalografia por Raios X , Éxons/genética , Humanos , Mutação , Neoplasias/química , Neoplasias/genética , Nucleotídeos , Motivo de Reconhecimento de RNA , Processamento de RNA/genética , Fator de Processamento U2AF/química , Dedos de Zinco
3.
Nucleic Acids Res ; 48(16): 9250-9261, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32813009

RESUMO

N 6-methylation of 2'-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2'-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4's in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3' splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.


Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , Processamento de RNA/genética , RNA Nuclear Pequeno/genética , Adenosina/genética , Catálise , Éxons/genética , Humanos , Metilação , Precursores de RNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Spliceossomos/genética
4.
Cytogenet Genome Res ; 160(5): 238-244, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32659759

RESUMO

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome.


Assuntos
Processamento Alternativo/genética , Grupo com Ancestrais do Continente Asiático/genética , Colágeno Tipo IV/genética , Mutação , Nefrite Hereditária/genética , Sítios de Splice de RNA/genética , Adulto , Linhagem Celular , Criança , Pré-Escolar , Simulação por Computador , Éxons/genética , Feminino , Hematúria/genética , Humanos , Masculino , Linhagem , Proteinúria/genética
5.
BMC Evol Biol ; 20(1): 85, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664916

RESUMO

BACKGROUND: ATP-binding cassette (ABC) transporters are involved in the active transportation of various endogenous or exogenous substances. Two ABCG2 gene subfamily members have been identified in birds. A detailed comparative study of the ABCG2 and ABCG2-like genes aid our understanding of their evolutionary history at the molecular level and provide a theoretical reference for studying the specific functions of ABCG2 and ABCG2-like genes in birds. RESULTS: We first identified 77 ABCG2/ABCG2-like gene sequences in the genomes of 41 birds. Further analysis showed that both the nucleic acid and amino acid sequences of ABCG2 and ABCG2-like genes were highly conserved and exhibited high homology in birds. However, significant differences in the N-terminal structure were found between the ABCG2 and ABCG2-like amino acid sequences. A selective pressure analysis showed that the ABCG2 and ABCG2-like genes were affected by purifying selection during the process of bird evolution. CONCLUSIONS: We believe that multiple members of the ABCG2 gene subfamily exist on chromosome 4 in the ancestors of birds. Over the long course of evolution, only the ABCG2 gene was retained on chromosome 4 in birds. The ABCG2-like gene on chromosome 6 might have originated from chromosome replication or fusion. The structural differences between the N terminus of ABCG2 protein and those of ABCG2-like proteins might lead to functional differences between the corresponding genes.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Aves/genética , Evolução Molecular , Homologia de Sequência de Aminoácidos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Cromossomos/genética , Sequência Conservada/genética , Éxons/genética , Regulação da Expressão Gênica , Genoma , Íntrons/genética , Família Multigênica , Fases de Leitura Aberta/genética , Fosforilação , Filogenia , Domínios Proteicos , Sítios de Splice de RNA/genética , Seleção Genética , Sintenia/genética
6.
Nature ; 582(7812): 438-442, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555469

RESUMO

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


Assuntos
Processamento Alternativo/genética , Regulação Viral da Expressão Gênica , HIV-1/genética , Mutação , Sítios de Splice de RNA/genética , RNA Viral/química , RNA Viral/genética , Algoritmos , Sequência de Bases , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Dobramento de RNA , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Ésteres do Ácido Sulfúrico , Termodinâmica
7.
RNA ; 26(10): 1389-1399, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522889

RESUMO

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


Assuntos
Regulação Alostérica/genética , Processamento Alternativo/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Spliceossomos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteômica/métodos , Precursores de RNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética
8.
Nature ; 584(7822): 619-623, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32581359

RESUMO

Autoimmune thyroid disease is the most common autoimmune disease and is highly heritable1. Here, by using a genome-wide association study of 30,234 cases and 725,172 controls from Iceland and the UK Biobank, we find 99 sequence variants at 93 loci, of which 84 variants are previously unreported2-7. A low-frequency (1.36%) intronic variant in FLT3 (rs76428106-C) has the largest effect on risk of autoimmune thyroid disease (odds ratio (OR) = 1.46, P = 2.37 × 10-24). rs76428106-C is also associated with systemic lupus erythematosus (OR = 1.90, P = 6.46 × 10-4), rheumatoid factor and/or anti-CCP-positive rheumatoid arthritis (OR = 1.41, P = 4.31 × 10-4) and coeliac disease (OR = 1.62, P = 1.20 × 10-4). FLT3 encodes fms-related tyrosine kinase 3, a receptor that regulates haematopoietic progenitor and dendritic cells. RNA sequencing revealed that rs76428106-C generates a cryptic splice site, which introduces a stop codon in 30% of transcripts that are predicted to encode a truncated protein, which lacks its tyrosine kinase domains. Each copy of rs76428106-C doubles the plasma levels of the FTL3 ligand. Activating somatic mutations in FLT3 are associated with acute myeloid leukaemia8 with a poor prognosis and rs76428106-C also predisposes individuals to acute myeloid leukaemia (OR = 1.90, P = 5.40 × 10-3). Thus, a predicted loss-of-function germline mutation in FLT3 causes a reduction in full-length FLT3, with a compensatory increase in the levels of its ligand and an increased disease risk, similar to that of a gain-of-function mutation.


Assuntos
Códon sem Sentido/genética , Predisposição Genética para Doença/genética , Ligantes , Mutação , Tireoidite Autoimune/genética , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Alelos , Doenças Autoimunes/genética , Bases de Dados Factuais , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa , Humanos , Islândia , Íntrons/genética , Leucemia Mieloide Aguda , Mutação com Perda de Função , Sítios de Splice de RNA/genética , Reino Unido
9.
Doc Ophthalmol ; 141(3): 217-226, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32333190

RESUMO

PURPOSE: We report a 15-month follow-up case on a Chinese patient with Oguchi disease associated with the multiple evanescent white dot syndrome (MEWDS). METHODS: The patient's clinical presentation and follow-up visits were documented via decimal best-corrected visual acuity, fundus photography, fundus autofluorescence (FAF) imaging, near-infrared FAF, spectral domain optical coherence tomography, Humphrey's visual fields, microperimetry, and multifocal electroretinography. We also performed whole exome sequencing for screening variation in the patient and her relatives. RESULTS: The patient had typical clinical characteristic of Oguchi disease, including night blindness, the Mizuo-Nakamura phenomenon (a golden yellow discoloration of the fundus that disappears in the prolonged dark adaptation [DA]) and typical full-field electroretinogram changes (nearly undetected b-wave in 0.01 and 0.03 ERGs that can partially recover only after prolonged DA). Aside from Oguchi disease, the patient was also diagnosed with the MEWDS based on clinical detections, including suddenly reduced visual acuity, appeared white dots, blurred ellipsoid zone and disrupted interdigitation zone, enlarged blind spot, and reduced macular sensitivity. A series of investigations revealed that along with the 15-month follow-up after onset, the visual acuity enhanced, the numerous white dots disappeared, and the macular structure returned to normal. Moreover, the novel homozygous splicing alteration c.181 + 1G > A was identified in the SAG gene. CONCLUSIONS: This work is the first long-term case study of a patient with Oguchi disease associated with the MEWDS. The recovery period of symptoms caused by the MEWDS was much longer than that in typical patients with MEWDS. Molecular genetics demonstrate that this is the first case of Oguchi disease caused by splicing alterations in the SAG gene.


Assuntos
Arrestina/genética , Oftalmopatias Hereditárias/diagnóstico , Oftalmopatias Hereditárias/genética , Cegueira Noturna/diagnóstico , Cegueira Noturna/genética , Sítios de Splice de RNA/genética , Síndromes do Ponto Branco/diagnóstico , Adulto , Adaptação à Escuridão , Eletrorretinografia , Oftalmopatias Hereditárias/fisiopatologia , Feminino , Seguimentos , Humanos , Cegueira Noturna/fisiopatologia , Linhagem , Reação em Cadeia da Polimerase , Processamento de RNA , Retina/fisiopatologia , Escotoma/diagnóstico , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Testes de Campo Visual , Campos Visuais/fisiologia , Síndromes do Ponto Branco/fisiopatologia , Sequenciamento Completo do Exoma
10.
RNA ; 26(8): 996-1005, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32312846

RESUMO

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.


Assuntos
Eritroblastos/fisiologia , Éxons/genética , Oligonucleotídeos Antissenso/genética , Processamento Alternativo/genética , Células Cultivadas , Humanos , Íntrons/genética , N-Acetilglucosaminiltransferases/genética , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA/genética
11.
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
12.
BMC Med Genet ; 21(1): 61, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32216767

RESUMO

BACKGROUND: Wolcott-Rallison Syndrome (WRS) is a rare autosomal recessive disease that is the most common cause of neonatal diabetes in consanguineous families. WRS is caused by various genetic alterations of the Eukaryotic Translation Initiation Factor 2-Alpha Kinase 3 (EIF2AK3) gene. METHODS: Genetic analysis of a consanguineous family where two children were diagnosed with WRS was performed by Sanger sequencing. The altered protein was investigated by in vitro cloning, expression and immunohistochemistry. RESULTS: The first cases in Hungary, - two patients in one family, where the parents were fourth-degree cousins - showed the typical clinical features of WRS: early onset diabetes mellitus with hyperglycemia, growth retardation, infection-induced multiple organ failure. The genetic background of the disease was a novel alteration in the EIF2AK3 gene involving the splice site of exon 11- intron 11-12 boundary: g.53051_53062delinsTG. According to cDNA sequencing this created a new splice site and resulted in a frameshift and the development of an early termination codon at amino acid position 633 (p.Pro627AspfsTer7). Based on in vitro cloning and expression studies, the truncated protein was functionally inactive. Immunohistochemistry revealed that the intact protein was absent in the islets of pancreas, furthermore insulin expressing cells were also dramatically diminished. Elevated GRP78 and reduced CHOP protein expression were observed in the liver. CONCLUSIONS: The novel genetic alteration causing the absence of the EIF2AK3 protein resulted in insufficient handling of severe endoplasmic reticulum stress, leading to liver failure and demise of the patients.


Assuntos
Diabetes Mellitus Tipo 1/genética , Epífises/anormalidades , Mutação INDEL , Osteocondrodisplasias/genética , Sítios de Splice de RNA/genética , eIF-2 Quinase/genética , Pré-Escolar , Consanguinidade , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/patologia , Estresse do Retículo Endoplasmático/genética , Epífises/patologia , Evolução Fatal , Feminino , Mutação da Fase de Leitura , Humanos , Hungria , Lactente , Falência Hepática/complicações , Falência Hepática/genética , Falência Hepática/patologia , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/patologia , Linhagem , Irmãos , Viroses/complicações , Viroses/patologia
13.
Nat Commun ; 11(1): 1438, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188845

RESUMO

While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.


Assuntos
Regulação para Baixo/genética , Íntrons/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Adulto , Processamento Alternativo/genética , Sequência de Bases , Humanos , Sequenciamento por Nanoporos , Isoformas de Proteínas/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Cell ; 180(1): 208-208.e1, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31951519

RESUMO

RNA splicing, the spliceosome-catalyzed process by which pre-messenger RNA (pre-mRNA) is processed to mature mRNA, is altered in a number of ways in cancer. Tumor-specific splicing alterations are created by mutations that disrupt splicing-regulatory elements within genes and impair splicing recognition or by altering the RNA-binding preferences of individual splicing factors. This SnapShot summarizes our current understanding of splicing-factor alterations in cancers. To view this SnapShot, open or download the PDF.


Assuntos
Processamento Alternativo/genética , Neoplasias/genética , Sítios de Splice de RNA/genética , Humanos , Mutação , Precursores de RNA/metabolismo , Processamento de RNA/genética , Processamento de RNA/fisiologia , Fatores de Processamento de RNA/genética , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo
15.
Doc Ophthalmol ; 141(1): 77-88, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31997113

RESUMO

PURPOSE: To report the clinical and genetic features of a 9-year-old female Japanese patient with Bardet-Biedl syndrome (BBS). METHODS: Genetic analysis using whole-exome sequencing (WES) was performed for the patient and her parents to identify disease-causing variants. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to investigate the impact of splice-site variants. Comprehensive ophthalmic and systemic examinations, including electroretinography (ERG), were performed. RESULTS: In the patient, WES identified novel compound heterozygous splice-site variants (c.124+2T>G and c.723+2T>G) in the BBS1 gene, and RT-PCR revealed skipping of exons 2 and 8 (p.N17AfsX56 and p.T198_K241del). Each parent had one of the variants. Ophthalmologically, the patient's decimal best-corrected visual acuity was 0.6 in the right eye and 0.4 in the left eye. Funduscopy revealed no apparent retinal degeneration or narrowed blood vessels in the periphery, but macular abnormalities were found on fundus autofluorescence imaging and optical coherence tomography images. Unexpectedly, non-recordable responses in rod ERG were found, with a non-recordable response of the right eye and an extremely reduced and delayed a-wave of the left eye in standard ERG, non-recordable responses in cone ERG, and extremely decreased responses in 30 Hz flicker ERG. Finally, the patient fulfilled four primary features of BBS diagnostic criteria: rod-cone dystrophy, polydactyly, central obesity, and learning disabilities, being diagnosed with BBS. CONCLUSIONS: This is the first report of a BBS patient with biallelic splice-site BBS1 variants in the Japanese population. Disparity between funduscopic and ERG findings may be a feature of BBS1-associated rod-cone dystrophy.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Síndrome de Bardet-Biedl/genética , Variação Genética/genética , Proteínas Associadas aos Microtúbulos/genética , Sítios de Splice de RNA/genética , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Síndrome de Bardet-Biedl/diagnóstico , Síndrome de Bardet-Biedl/fisiopatologia , Criança , Análise Mutacional de DNA , Eletrorretinografia , Éxons/genética , Feminino , Humanos , Japão/epidemiologia , Mutação , Oftalmoscopia , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Tomografia de Coerência Óptica , Sequenciamento Completo do Exoma
16.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906439

RESUMO

CDC14A encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene CDC14A, respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that CDC14A is associated with a rare form of hereditary hearing loss.


Assuntos
Perda Auditiva Neurossensorial/genética , Proteínas Tirosina Fosfatases/genética , Adulto , Exoma/genética , Feminino , Estudos de Associação Genética , Humanos , Irã (Geográfico) , Mutação com Perda de Função , Masculino , Paquistão , Linhagem , Proteínas Tirosina Fosfatases/metabolismo , Sítios de Splice de RNA/genética , Processamento de RNA
17.
Nucleic Acids Res ; 48(5): 2676-2693, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31943118

RESUMO

Besides analyses of specific alternative splicing (AS) variants, little is known about AS regulatory pathways and programs involved in anticancer drug resistance. Doxorubicin is widely used in breast cancer chemotherapy. Here, we identified 1723 AS events and 41 splicing factors regulated in a breast cancer cell model of acquired resistance to doxorubicin. An RNAi screen on splicing factors identified the little studied ZRANB2 and SYF2, whose depletion partially reversed doxorubicin resistance. By RNAi and RNA-seq in resistant cells, we found that the AS programs controlled by ZRANB2 and SYF2 were enriched in resistance-associated AS events, and converged on the ECT2 splice variant including exon 5 (ECT2-Ex5+). Both ZRANB2 and SYF2 were found associated with ECT2 pre-messenger RNA, and ECT2-Ex5+ isoform depletion reduced doxorubicin resistance. Following doxorubicin treatment, resistant cells accumulated in S phase, which partially depended on ZRANB2, SYF2 and the ECT2-Ex5+ isoform. Finally, doxorubicin combination with an oligonucleotide inhibiting ECT2-Ex5 inclusion reduced doxorubicin-resistant tumor growth in mouse xenografts, and high ECT2-Ex5 inclusion levels were associated with bad prognosis in breast cancer treated with chemotherapy. Altogether, our data identify AS programs controlled by ZRANB2 and SYF2 and converging on ECT2, that participate to breast cancer cell resistance to doxorubicin.


Assuntos
Processamento Alternativo/genética , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo/efeitos dos fármacos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNA/genética , Fase S/efeitos dos fármacos , Spliceossomos/metabolismo
18.
Nat Commun ; 11(1): 137, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919425

RESUMO

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.


Assuntos
Processamento Alternativo/genética , Biologia Computacional/métodos , Análise de Dados , Células Fotorreceptoras/citologia , Sítios de Splice de RNA/genética , Animais , Linhagem Celular Tumoral , Expressão Gênica/genética , Células Hep G2 , Ribonucleoproteínas Nucleares Heterogêneas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Hepáticas/genética , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Retina/citologia , Análise de Sequência de RNA/métodos
19.
Nucleic Acids Res ; 48(2): 802-816, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31802121

RESUMO

Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15-25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.


Assuntos
Hibridização Genética , Oligonucleotídeos Antissenso/genética , Sítios de Splice de RNA/genética , Processamento de RNA/genética , Sítios de Ligação/genética , Linhagem Celular , Éxons/genética , Humanos , Hibridização de Ácido Nucleico/genética , Precursores de RNA/genética , RNA Mensageiro/genética
20.
J Dairy Sci ; 103(1): 607-612, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733857

RESUMO

A genome scan for homozygous haplotype deficiency coupled with whole-genome sequence data analysis is a very effective method to identify embryonic lethal mutations in cattle. Among other factors, the power of the approach depends on the availability of a greater amount of genotyping and sequencing data. In the present study, we analyzed the largest known panel of Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes, comprising 401,896 Holstein animals, and we report the mapping of a new embryonic lethal haplotype on chromosome 27, called HH7. We fine mapped the locus in a 2.0-Mb interval using an identical-by-descent approach and analyzed genome sequence data from 4 carrier and 143 noncarrier Holstein bulls to identify the causative mutation. We detected a strong candidate variant in the gene encoding centromere protein U (CENPU), a centromere component essential for proper chromosome segregation during mitosis. The mutant allele is a deletion of 4 nucleotides located at position +3 to +6 bp after the splicing donor site of exon 11. Cross-species nucleotide alignment revealed that the nucleotide at position +3 is entirely conserved among vertebrates, suggesting that it plays an important role in the regulation of CENPU splicing. For verification, we genotyped the candidate variant in 232,775 Holstein individuals and did not observe any homozygotes, whereas 16 were expected (Poisson P-value = 1.1 × 10-7; allele frequency = 0.8%). In addition, genotyping of 250,602 animals from 19 additional breeds revealed that the mutant allele is restricted to animals of Holstein descent. Finally, we estimated the effect of the candidate variant on 2 fertility traits in at-risk mating (i.e., between carrier bulls and daughters of carrier bulls) versus non-risk mating. In agreement with a recessive lethal inheritance pattern, we observed a marked reduction in both conception rate and 56-d nonreturn rate in heifers and cows. The effect on 56-d nonreturn rate suggests that a substantial proportion of homozygous mutants die before 35 d after insemination, which is consistent with the early embryonic death previously reported in CENPU-/- mouse embryos. In conclusion, we demonstrate that with more than 400,000 genotypes, we can map very rare recessive lethal mutations segregating at a frequency below 1% in the population. We recommend performing new analyses regularly as data are accumulating.


Assuntos
Centrômero/genética , Perda do Embrião/veterinária , Histonas/genética , Mutação , Sítios de Splice de RNA/genética , Alelos , Animais , Bovinos , Perda do Embrião/genética , Feminino , Fertilidade/genética , Fertilização , Genótipo , Haplótipos , Homozigoto , Fenótipo
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