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1.
J Clin Pathol ; 72(11): 778-782, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473630

RESUMO

SF3B1 is the largest subunit of the Spliceosome Factor 3b (SF3B) complex and part of the U2 small nuclear ribosomal protein. It functions as an important part of spliceosomal assembly, converting precursor messenger RNA (mRNA) to mRNA ready for ribosomal translation. Mutations of SF3B1 are commonly seen in myelodysplastic syndromes with ring sideroblasts (MDS-RS)and MDS/myeloproliferative neoplasm (MPN-RS-T). These mutations are typically heterozygous missense substitutions, of which, 55% involve K700E. MDS-RS and MDS/MPN-RS-T usually carry a more favourable prognosis than other subtypes of MDS. SF3B1 itself does not influence survival in these conditions, but does correlate with increased thrombotic risk. Mutated SF3B1 is present in 9%-15% of chronic lymphocytic leukaemia cases and on its own correlates with improved responsiveness to ibrutinib, but is associated with additional adverse genetic abnormalities including TP53 and ATM mutations, which traditionally confer adverse outcomes.


Assuntos
Biomarcadores Tumorais/genética , Eritroblastos/patologia , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Predisposição Genética para Doença , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/terapia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/terapia , Fenótipo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Prognóstico , Conformação Proteica , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Relação Estrutura-Atividade
2.
Rinsho Ketsueki ; 60(8): 915-919, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31484889

RESUMO

A 83-year-old female patient was admitted to our hospital due to hematological manifestation of juvenile granulocytes and macrocytic anemia. Bone marrow (BM) examination revealed erythroid dysplasia and cytoplasmic blasts, and hence the patient was diagnosed with myelodysplastic syndrome with ring sideroblasts and with single lineage dysplasia (MDS-RS-SLD). Erythrocyte transfusion was performed as a supportive therapy, and there was a gradual increase in the number of blood cells. Therefore, BM re-examination was performed and it was confirmed that the number of megakaryocytes increased, so the patient's condition was determined as myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis (MDS/MPN with RS-T). Incidentally, gene mutation analysis showed CALR gene mutation. Thereafter, administration of hydroxycarbamide and anagrelide did not show adverse events and complications, and a good blood count control was obtained. Furthermore, it was also confirmed that an SF3B1 gene mutation is highly positive in MDS-RS. There was no report on CALR-mutant MDS/MPN in Japan, and it is a rare disease overseas.


Assuntos
Calreticulina/genética , Neoplasias Hematológicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Trombocitose , Idoso de 80 Anos ou mais , Feminino , Humanos , Japão , Mutação , Trombocitose/genética
3.
BMC Biol ; 17(1): 56, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311534

RESUMO

BACKGROUND: Adaptive responses to stress are essential for cell and organismal survival. In metazoans, little is known about the impact of environmental stress on RNA homeostasis. RESULTS: By studying the regulation of a cadmium-induced gene named numr-1 in Caenorhabditis elegans, we discovered that disruption of RNA processing acts as a signal for environmental stress. We find that NUMR-1 contains motifs common to RNA splicing factors and influences RNA splicing in vivo. A genome-wide screen reveals that numr-1 is strongly and specifically induced by silencing of genes that function in basal RNA metabolism including subunits of the metazoan integrator complex. Human integrator processes snRNAs for functioning with splicing factors, and we find that silencing of C. elegans integrator subunits disrupts snRNA processing, causes aberrant pre-mRNA splicing, and induces the heat shock response. Cadmium, which also strongly induces numr-1, has similar effects on RNA and the heat shock response. Lastly, we find that heat shock factor-1 is required for full numr-1 induction by cadmium. CONCLUSION: Our results are consistent with a model in which disruption of integrator processing of RNA acts as a molecular damage signal initiating an adaptive stress response mediated by heat shock factor-1. When numr-1 is induced via this pathway in C. elegans, its function in RNA metabolism may allow it to mitigate further damage and thereby promote tolerance to cadmium.


Assuntos
Cádmio/toxicidade , Caenorhabditis elegans/fisiologia , Regulação da Expressão Gênica , Resposta ao Choque Térmico/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Processamento de RNA , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Resposta ao Choque Térmico/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Estresse Fisiológico
4.
Retrovirology ; 16(1): 12, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036027

RESUMO

BACKGROUND: The different interactions between viral proteins and cellular host proteins are required for efficient replication of HIV-1. Various reports implicated host cellular proteins as a key factor that either interact directly with HIV-1 integrase (IN) or get involved in the integration process of virus resulting in the modulation of integration step. Polypyrimidine tract binding protein and associated splicing factor (PSF) has diverse functions inside the cell such as transcriptional regulation, DNA repair, acts as nucleic acids binding protein and regulate replication and infectivity of different viruses. RESULTS: The protein binding study identified the association of host protein PSF with HIV-1 integrase. The siRNA knockdown (KD) of PSF resulted in increased viral replication in TZM-bl cells, suggesting PSF has negative influence on viral replication. The quantitative PCR of virus infected PSF knockdown TZM-bl cells showed more integrated DNA and viral cDNA as compared to control cells. We did not observe any significant difference between the amount of early reverse transcription products as well as infectivity of virus in the PSF KD and control TZM-bl cells. Molecular docking study supported the argument that PSF hinders the binding of viral DNA with IN. CONCLUSION: In an attempt to study the host interacting protein of IN, we have identified a new interacting host protein PSF which is a splicing factor and elucidated its role in integration and viral replication. Experimental as well as in silico analysis inferred that the host protein causes not only change in the integration events but also targets the incoming viral DNA or the integrase-viral DNA complex. The role of PSF was also investigated at early reverse transcript production as well as late stages. The PSF is causing changes in integration events, but it does not over all make any changes in the virus infectivity. MD trajectory analyses provided a strong clue of destabilization of Integrase-viral DNA complex occurred due to PSF interaction with the conserved bases of viral DNA ends that are extremely crucial contact points with integrase and indispensable for integration. Thus our study emphasizes the negative influence of PSF on HIV-1 replication.


Assuntos
DNA Viral/metabolismo , Integrase de HIV/metabolismo , Interações entre Hospedeiro e Microrganismos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/metabolismo , Replicação Viral , DNA Viral/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Integrase de HIV/genética , HIV-1/fisiologia , Humanos , Simulação de Acoplamento Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica , Processamento de RNA , Fatores de Processamento de RNA/genética , RNA Interferente Pequeno , Transcrição Reversa , Integração Viral
5.
Medicine (Baltimore) ; 98(21): e15743, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31124956

RESUMO

BACKGROUND: Gene mutations with important prognostic role have been identified in patients with myelodysplastic syndrome (MDS). We performed a meta-analysis to investigate the effects of RNA splicing machinery gene mutations on prognosis of MDS patients. METHODS: We searched English database including PubMed, Embase, Cochrane Library for literatures published within recent 10 years on the effect of RNA splicing machinery genes in MDS. Revman version 5.2 software was used for all the statistical processing. We calculated risk ratio and 95% confidence interval (CI) of continuous variables, and find hazard ratio (HR) and 95% CI of time-to-event data. RESULTS: We included 19 studies enrolling 4320 patients. There is a significant superior overall survival (OS) in splicing factor 3b, subunit 1 (SF3B1)-mutation group compared to unmutated group (HR = 0.58, 95% CI: 0.5-0.67, P < .00001); OS decreased significantly in serine/arginine-rich splicing factor 2/ U2 auxiliary factor protein 1 (SRSF2/U2AF1) mutation group compared to unmutated group, (HR = 1.62, 95% CI: 1.34-1.97, P < .00001 and HR = 1.61, 95% CI: 1.35-1.9, P < .00001, respectively). In terms of leukemia-free survival (LFS), the group with SF3B1 mutation had better outcome than unmutated group, HR = 0.63 (95% CI: 0.53-0.75, P < .00001). Other RNA splicing gene mutation group showed significant poor LFS than unmutated groups, (HR = 1.89, 95% CI: 1.6-2.23, P < .00001; HR = 2.77, 95% CI: 2.24-3.44, P < .00001; HR = 1.48, 95% CI: 1.08-2.03, P < .00001; for SRSF2, U2AF1, and zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 [ZRSR2], respectively). As for subgroup of low- or intermediate-1-IPSS risk MDS, SRSF2, and U2AF1 mutations were related to poor OS. (HR = 1.83, 95% CI: 1.43-2.35, P < .00001; HR = 2.11, 95% CI: 1.59-2.79, P < .00001 for SRSF2 and U2AF1, respectively). SRSF2 and U2AF1 mutations were strongly associated with male patients. SF3B1 mutation was strongly associated with disease staging. CONCLUSION: This meta-analysis indicates a positive effect of SF3B1 and an adverse prognostic effect of SRSF2, U2AF1, and ZRSR2 mutations in patients with MDS. Mutations of RNA splicing genes have important effects on the prognosis of MDS.


Assuntos
Síndromes Mielodisplásicas/genética , Processamento de RNA/genética , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Prognóstico , Fatores de Processamento de RNA/genética , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Índice de Gravidade de Doença , Fatores Sexuais , Fator de Processamento U2AF/genética , Análise de Sobrevida
6.
DNA Cell Biol ; 38(7): 627-638, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31025877

RESUMO

Head and neck squamous cell carcinoma (HNSC) is a common malignancy with high mortality and poor prognosis. Alternative splicing (AS) is a transcriptional regulation mechanism that generates multiple transcripts from same genes, and aberrant AS signatures of cancers can be predictive for prognosis. We identified the survival-related AS events and splicing factors (SFs) from the RNA sequencing data and the corresponding clinical information of an HNSC cohort downloaded from The Cancer Genome Atlas (TCGA) and SpliceSeq. The independent prognostic predictors were assessed by Cox proportional regression analysis, and the regulatory network of SFs and AS events was analyzed by Spearman's test and constructed. A total of 4626 survival-related AS events in 3280 genes were identified, and most were protective factors. Among the different types of splicing events, exon skip was the most frequent. The prognostic models were constructed for each type of AS, and the area under the curve of the receiver operating characteristic curve of the combined prognostic model was 0.765, indicating good predictive performance. Finally, a correlation network between SF and AS events was constructed. We identified prognostic predictors based on AS events that stratified HNSC patients into the high- and low-risk groups, and revealed splicing networks that provide insights into the underlying mechanisms.


Assuntos
Processamento Alternativo , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Fatores de Processamento de RNA/genética , RNA Mensageiro/genética , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo
7.
Biomolecules ; 9(4)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939845

RESUMO

Although inhibition of the androgen⁻androgen receptor (AR) axis effectively represses the growth of prostate cancer, most of all cases eventually become castration-resistant prostate cancers (CRPCs). Enhancement of the expression of AR and its variants along with the downstream signals is important for disease progression. AR-V7, a constitutive active form of AR, is generated as a result of RNA splicing. RNA splicing creates multiple transcript variants from one pre-messenger RNA (mRNA) by removing introns/exons to allow mRNA translation. The molecular mechanisms leading to marked increases of AR and generation of AR-V7 have been unclear. However, recent papers highlighted the roles of RNA splicing factors which promote AR expression and production of variants. Notably, a broad range of splicing components were aberrantly regulated in CRPC tissues. Interestingly, expression of various spliceosome genes is enhanced by RNA-binding protein splicing factor proline- and glutamine-rich (PSF/SFPQ), leading to changes in the expression of AR transcript variants. Moreover, inhibition of several splicing factors repressed tumor growth in vivo. Altered expression of splicing factors is correlated to biochemical recurrence in prostate cancer patients. Thus, these findings suggest that splicing factors would be a potential therapeutic target. This review focuses on the emerging roles of splicing factors in prostate cancer progression and AR signaling.


Assuntos
Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Processamento de RNA/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Animais , Humanos , Masculino , Neoplasias da Próstata/genética , Fatores de Processamento de RNA/genética , Transdução de Sinais/genética
8.
Cancer Sci ; 110(6): 2004-2013, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980774

RESUMO

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed.


Assuntos
Autoanticorpos/imunologia , Neoplasias Esofágicas/imunologia , Carcinoma de Células Escamosas do Esôfago/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Processamento de RNA/imunologia , Proteínas Repressoras/imunologia , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/diagnóstico , Carcinoma de Células Escamosas do Esôfago/genética , Éxons/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de RNA , Fatores de Processamento de RNA/genética , Curva ROC , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/imunologia
9.
Nat Commun ; 10(1): 1590, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962446

RESUMO

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Oligonucleotídeos/farmacologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Músculo Esquelético/crescimento & desenvolvimento , Degradação do RNAm Mediada por Códon sem Sentido , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/antagonistas & inibidores , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/metabolismo , Sequências de Repetição em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia , Peixe-Zebra/genética
10.
Asian Pac J Cancer Prev ; 20(4): 1215-1221, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31030497

RESUMO

Background: The frequency and pattern of mutation in SF3B1 and SRSF2 RNA splicing machinery genes were found to vary among myelodysplastic syndrome (MDS) patients in different populations. There have been less reports of incidence of these gene mutations in Thailand especially in upper northern Thailand. This study therefore had aims to investigate the frequency and pattern of mutation in mutational hotspot of SF3B1 and SRSF2 genes among MDS patients in upper northern Thailand and to investigate the clinical features associated with the mutations. Methods: Fifty-five MDS patients who underwent treatment at Maharaj Nakorn Chiang Mai Hospital participated in this study. The detection of SF3B1 and SRSF2 hotspot mutations was carried out using polymerase chain reaction followed by Sanger sequencing. In addition, clinical features of individual patients with these gene mutations were also investigated. Results: SF3B1 mutations (SF3B1mut) were found in 9 patients (16.4%) including E622D (1/9), R625C (1/9), H662Q (1/9), K700E (5/9), and Q699H co-mutation with K700E (1/9). SRSF2 mutations (SRSF2mut) were found in 4 patients (7.3%) which included P95H (3/4) and P95L (1/4). The SF3B1mut was associated with lower hemoglobin levels (p = 0.023) and higher platelet counts (p = 0.047) when compared with MDS patients without SF3B1mut, while SRSF2mut tended to occur in patients with a higher percentage of bone marrow blasts (p = 0.074). Conclusion: The findings confirmed the difference in frequency of SF3B1 and SRSF2 mutations among different populations. Specifically, we found a co-mutation of Q699H and K700E that has not been previously reported in MDS patients in the COSMIC database. It was also found that SF3B1mut was strongly associated with low hemoglobin level, and high platelet counts whereas SRSF2mut was mostly clustered in MDS with excess blasts subsequently increasing the probability of progression to acute myeloid leukemia.


Assuntos
Biomarcadores Tumorais/genética , Mutação , Síndromes Mielodisplásicas/epidemiologia , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Tailândia/epidemiologia
11.
Nat Ecol Evol ; 3(4): 691-701, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833759

RESUMO

The mechanisms by which entire programmes of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates, and are critical for proper brain development and function. Here, we discover neural microexon programmes in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized 'enhancer of microexons' (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralogue Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programmes that qualitatively expanded the neuronal molecular complexity in bilaterians.


Assuntos
Éxons , Neurônios , Fatores de Processamento de RNA/genética , Processamento Alternativo , Animais , Artrópodes , Drosophila melanogaster , Evolução Molecular , Humanos , Anfioxos , Camundongos , Domínios Proteicos , Peixe-Zebra
12.
Planta ; 249(6): 1997-2014, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904945

RESUMO

MAIN CONCLUSION: The work offers a comprehensive evaluation on the phylogenetics and conservation of splicing patterns of the plant SPF30 splicing factor gene family. In eukaryotes, one pre-mRNA can generate multiple mRNA transcripts by alternative splicing (AS), which expands transcriptome and proteome diversity. Splicing factor 30 (SPF30), also known as survival motor neuron domain containing protein 1 (SMNDC1), is a spliceosomal protein that plays an essential role in spliceosomal assembly. Although SPF30 genes have been well characterised in human and yeast, little is known about their homologues in plants. Here, we report the genome-wide identification and phylogenetic analysis of SPF30 genes in the plant kingdom. In total, 82 SPF30 genes were found in 64 plant species from algae to land plants. Alternative transcripts were found in many SPF30 genes and splicing profile analysis revealed that the second intron in SPF30 genome is frequently associated with AS events and contributed to the birth of novel exons in a few SPF30 members. In addition, different conserved sequences were observed at these putative splice sites among moss, monocots and dicots, respectively. Our findings will facilitate further functional characterization of plant SPF30 genes as putative splicing factors.


Assuntos
Processamento Alternativo/genética , Plantas/genética , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Evolução Biológica , Sequência Conservada , Éxons/genética , Íntrons/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , Spliceossomos/genética , Spliceossomos/metabolismo
13.
Genetics ; 212(1): 111-124, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30898770

RESUMO

Selection of suppressor mutations that correct growth defects caused by substitutions in an RNA or protein can reveal functionally important molecular structures and interactions in living cells. This approach is particularly useful for the study of complex biological pathways involving many macromolecules, such as premessenger RNA (pre-mRNA) splicing. When a sufficiently large number of suppressor mutations is obtained and structural information is available, it is possible to generate detailed models of molecular function. However, the laborious and expensive task of identifying suppressor mutations in whole-genome selections limits the utility of this approach. Here I show that a custom targeted sequencing panel can greatly accelerate the identification of suppressor mutations in the Saccharomyces cerevisiae genome. Using a panel that targets 112 genes encoding pre-mRNA splicing factors, I identified 27 unique mutations in six protein-coding genes that each overcome the cold-sensitive block to spliceosome activation caused by a substitution in U4 small nuclear RNA. When mapped to existing structures of spliceosomal complexes, the identified suppressors implicate specific molecular contacts between the proteins Brr2, Prp6, Prp8, Prp31, Sad1, and Snu114 as functionally important in an early step of catalytic activation of the spliceosome. This approach shows great promise for elucidating the allosteric cascade of molecular interactions that direct accurate and efficient pre-mRNA splicing and should be broadly useful for understanding the dynamics of other complex biological assemblies or pathways.


Assuntos
Regulação Alostérica , Fatores de Processamento de RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Supressão Genética , Análise Mutacional de DNA , Regulação Fúngica da Expressão Gênica , Conformação Proteica , Fatores de Processamento de RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética
14.
Nucleic Acids Res ; 47(7): 3450-3466, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30657957

RESUMO

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Genoma Humano/genética , Instabilidade Genômica/genética , Células Hep G2 , Humanos , Processamento de RNA/genética , Troca de Cromátide Irmã/genética
15.
Nucleic Acids Res ; 47(7): 3667-3679, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30698802

RESUMO

RNA secondary structures have been increasingly recognized to play an important regulatory role in post-transcriptional gene regulation. We recently showed that RNA G-quadruplexes, which serve as cis-elements to recruit splicing factors, play a critical role in regulating alternative splicing during the epithelial-mesenchymal transition. In this study, we performed a high-throughput screen using a dual-color splicing reporter to identify chemical compounds capable of regulating G-quadruplex-dependent alternative splicing. We identify emetine and its analog cephaeline as small molecules that disrupt RNA G-quadruplexes, resulting in inhibition of G-quadruplex-dependent alternative splicing. Transcriptome analysis reveals that emetine globally regulates alternative splicing, including splicing of variable exons that contain splice site-proximal G-quadruplexes. Our data suggest the use of emetine and cephaeline for investigating mechanisms of G-quadruplex-associated alternative splicing.


Assuntos
Processamento Alternativo/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Processamento de RNA/efeitos dos fármacos , RNA/química , Processamento Alternativo/genética , Emetina/farmacologia , Éxons/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA/efeitos dos fármacos , Processamento de RNA/genética , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Bibliotecas de Moléculas Pequenas/farmacologia
16.
Hum Genet ; 138(2): 199-210, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30671673

RESUMO

In this study, we investigated low-frequency and rare variants associated with blood pressure (BP) by focusing on a linkage region on chromosome 16p13. We used whole genome sequencing (WGS) data obtained through the NHLBI Trans-Omics for Precision Medicine (TOPMed) program on 395 Cleveland Family Study (CFS) European Americans (CFS-EA). By analyzing functional coding variants and non-coding rare variants with CADD score > 10 residing within the chromosomal region in families with linkage evidence, we observed 25 genes with nominal statistical evidence (burden or SKAT p < 0.05). One of the genes is RBFOX1, an evolutionarily conserved RNA-binding protein that regulates tissue-specific alternative splicing that we previously reported to be associated with BP using exome array data in CFS. After follow-up analysis of the 25 genes in ten independent TOPMed studies with individuals of European, African, and East Asian ancestry, and Hispanics (N = 29,988), we identified variants in SLX4 (p = 2.19 × 10-4) to be significantly associated with BP traits when accounting for multiple testing. We also replicated the associations previously reported for RBFOX1 (p = 0.007). Follow-up analysis with GTEx eQTL data shows SLX4 variants are associated with gene expression in coronary artery, multiple brain tissues, and right atrial appendage of the heart. Our study demonstrates that linkage analysis of family data can provide an efficient approach for detecting rare variants associated with complex traits in WGS data.


Assuntos
Pressão Sanguínea/genética , Cromossomos Humanos Par 16/genética , Exoma , Ligação Genética , Variação Genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Processamento Alternativo/genética , Feminino , Seguimentos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Fatores de Processamento de RNA/genética , Recombinases/genética
17.
Crit Rev Oncol Hematol ; 133: 74-83, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30661660

RESUMO

SF3B1 gene mutations are the most frequent mutations found in myelodysplastic syndromes (MDS), and the prognostic implication of these mutations remains controversial. We conducted a meta-analysis of studies assessing the prognostic impact and clinical feature of SF3B1 mutations in MDS patients. The overall hazard ratio for overall survival (OS) was 0.90 (95% confidence interval 0.60-1.35, P = 0.61) in MDS patients with SF3B1 mutations compared to those without. Lower leukemia-free survival was associated with SF3B1 mutations. Subgroup analyses showed that Asian cohorts and Illumina HiSeq 2000 methods were significantly associated with OS. Furthermore, SF3B1 mutations were significantly correlated with a lower level of blast cells and a high level of platelet counts and bone marrow ring sideroblasts. Thus, the current meta-analysis suggests that SF3B1 mutations have no significant impact on the OS of MDS patients, and the hematologic parameters of SF3B1 mutations identify a distinct subset of MDS patients with homogeneous features.


Assuntos
Mutação , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Biomarcadores Tumorais/genética , Humanos , Síndromes Mielodisplásicas/mortalidade , Síndromes Mielodisplásicas/terapia , Valor Preditivo dos Testes , Prognóstico , Análise de Sobrevida
18.
Nucleic Acids Res ; 47(4): e24, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30590765

RESUMO

Chimeric RNAs generated by cis-splicing between adjacent genes (cis-SAGe) are increasingly recognized as a widespread phenomenon. These chimeric messenger RNAs are present in normal human cells, and are also detected in various cancers. The mechanisms for how this group of chimeras is formed are not yet clear, in part due to the lack of a tractable system for their experimental investigation. Here we developed a fast, easy and versatile cell-based reporter system to identify regulators of cis-SAGe. The reporter, consisting of four main cassettes, simultaneously measures the effects of a candidate regulator on cis-SAGe and canonical splicing. Using this cell-based assay, we screened 102 candidate factors involved in RNA pol II cleavage and termination, elongation, splicing, alternative splicing and R-loop formation. We discovered that two factors, SRRM1 and SF3B1, affect not only cis-SAGe chimeras, but also other types of chimeric RNAs in a genome-wide fashion. This system can be used for studying trans-acting factors and cis-acting sequence elements and factors, as well as for screening small molecule inhibitors.


Assuntos
Antígenos Nucleares/genética , Regulação da Expressão Gênica/genética , Proteínas Associadas à Matriz Nuclear/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Fusão Gênica/genética , Genes Reporter/genética , Genoma Humano/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Clivagem do RNA/genética , RNA Polimerase II/genética
19.
EBioMedicine ; 39: 215-225, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30581150

RESUMO

INTRODUCTION: Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity. METHODS: Tissue-microarrays of 64 DMPM specimens were subjected to immunohistochemical assessment of SF3B1 expression and correlation to clinical outcome. Two primary cell cultures were used for gene expression profiling and in vitro screening of SF3b modulators. Drug-induced splicing alterations affecting downstream cellular pathways were detected through RNA sequencing. Ultimately, we established bioluminescent orthotopic mouse models to test the efficacy of splicing modulation in vivo. RESULTS: Spliceosomal genes are differentially upregulated in DMPM cells compared to normal tissues and high expression of SF3B1 correlated with poor clinical outcome in univariate and multivariate analysis. SF3b modulators (Pladienolide-B, E7107, Meayamycin-B) showed potent cytotoxic activity in vitro with IC50 values in the low nanomolar range. Differential splicing analysis of Pladienolide-B-treated cells revealed abundant alterations of transcripts involved in cell cycle, apoptosis and other oncogenic pathways. This was validated by RT-PCR and functional assays. E7107 demonstrated remarkable in vivo antitumor efficacy, with significant improvement of survival rates compared to vehicle-treated controls. CONCLUSIONS: SF3B1 emerged as a novel potential prognostic factor in DMPM. Splicing modulators markedly impair cancer cell viability, resulting also in potent antitumor activity in vivo. Our data designate splicing as a promising therapeutic target in DMPM.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neoplasias Peritoneais/tratamento farmacológico , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Processamento de RNA/efeitos dos fármacos , Análise Serial de Tecidos/métodos , Idoso , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Macrolídeos/administração & dosagem , Macrolídeos/farmacologia , Masculino , Mesotelioma/genética , Mesotelioma/metabolismo , Camundongos , Pessoa de Meia-Idade , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Fosfoproteínas/genética , Piranos/administração & dosagem , Piranos/farmacologia , Fatores de Processamento de RNA/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Biochem Biophys Res Commun ; 509(2): 384-389, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30594394

RESUMO

Alternative splicing (AS) is dysregulated in Type 1 diabetic (T1D) hearts but mechanisms responsible are unclear. Here, we provide evidence that the RNA binding protein (RBP) PTBP1 is modulated in adult T1D hearts contributing to AS changes. We show that a spliced variant of PTBP1 that is highly expressed in normal newborn mouse hearts is aberrantly expressed in adult T1D mouse hearts. Comparing known PTBP1-target datasets to our T1D mouse transcriptome datasets, we discovered a group of genes with PTBP1 binding sites in their pre-mRNAs that are differentially spliced in T1D mouse hearts. We demonstrated that inducible expression of diabetes-induced PTBP1 spliced variant has less repressive splicing function. Notably, PTBP1 regulates AS of some of its targets antagonistically to RBFOX2. In sum, our results indicate that diabetic conditions disrupt developmental regulation of PTBP1 leading to differential AS of PTBP1 target genes. Identification of PTBP1 and PTBP1-regulated RNA networks can provide RNA-based therapies for the treatment of diabetes cardiac complications.


Assuntos
Processamento Alternativo , Diabetes Mellitus Experimental/genética , Cardiomiopatias Diabéticas/genética , Regulação da Expressão Gênica no Desenvolvimento , Ribonucleoproteínas Nucleares Heterogêneas/genética , Miocárdio/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Animais , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Cardiomiopatias Diabéticas/induzido quimicamente , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Perfilação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Miocárdio/patologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Transdução de Sinais , Estreptozocina , Ativação Transcricional
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