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Immunotherapy harnesses neoantigens encoded within the human genome, but their therapeutic potential is hampered by low expression, which may be controlled by the nonsense-mediated mRNA decay (NMD) pathway. This study investigates the impact of UPF1-knockdown on the expression of non-canonical/mutant proteins, employing proteogenomic to explore UPF1 role within the NMD pathway. Additionally, we conducted a comprehensive pan-cancer analysis of UPF1 expression and evaluated UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue in-vivo. Our findings reveal that UPF1-knockdown leads to increased translation of non-canonical/mutant proteins, particularly those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated transcript biotypes. Moreover, our analysis demonstrates elevated UPF1 expression in various cancer types, with notably heightened protein levels in patient-derived TNBC tumors compared to adjacent tissues. This study elucidates UPF1 role in mitigating transcriptional noise by degrading transcripts encoding non-canonical/mutant proteins. Targeting this mechanism may reveal a new spectrum of neoantigens accessible to the antigen presentation pathway. Our novel findings provide a strong foundation for the development of therapeutic strategies aimed at targeting UPF1 or modulating the NMD pathway.
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BACKGROUND: Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered 844 lncRNAs that were genetically linked to breast cancer through genome-wide association studies (GWAS). Here, we show that a subset of these lncRNAs alter breast cancer risk by modulating cell proliferation, and provide evidence that a reduced expression on one lncRNA increases breast cancer risk through aberrant DNA replication and repair. METHODS: We performed pooled CRISPR-Cas13d-based knockdown screens in breast cells to identify which of the 844 breast cancer-associated lncRNAs alter cell proliferation. We selected one of the lncRNAs that increased cell proliferation, KILR, for follow-up functional studies. KILR pull-down followed by mass spectrometry was used to identify binding proteins. Knockdown and overexpression studies were performed to assess the mechanism by which KILR regulates proliferation. RESULTS: We show that KILR functions as a tumor suppressor, safeguarding breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. Mechanistically, KILR sequesters RPA1, a subunit of the RPA complex required for DNA replication and repair. Reduced KILR expression promotes breast cancer cell proliferation by increasing the available pool of RPA1 and speed of DNA replication. Conversely, KILR overexpression promotes apoptosis in breast cancer cells, but not normal breast cells. CONCLUSIONS: Our results confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.
Assuntos
Neoplasias da Mama , Sistemas CRISPR-Cas , Proliferação de Células , Reparo do DNA , Replicação do DNA , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica AmplaRESUMO
Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Netrinas/genética , Alelos , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica/métodos , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Netrinas/metabolismo , Fenótipo , Locos de Características Quantitativas , RiscoRESUMO
Background: Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. Methods: To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. Results: MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Conclusion: Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.
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CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.
Assuntos
Acetilcolinesterase/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Proteínas de Helminto/metabolismo , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/metabolismo , Acetilcolinesterase/genética , Animais , Feminino , Proteínas de Helminto/genética , Camundongos , Schistosoma mansoni/genética , Esquistossomose mansoni/genéticaRESUMO
Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 11/genética , Ciclina D1/genética , Reparo do DNA/genética , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Elementos Facilitadores Genéticos/genética , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença/genética , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Interferente Pequeno/genéticaRESUMO
A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.
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Cromossomos Humanos Par 14/genética , Neoplasias do Endométrio/genética , Fosfatidilinositol 3-Quinases/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Uterinas/genética , Fator de Transcrição YY1/metabolismo , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Risco , Transdução de Sinais , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Fator de Transcrição YY1/genéticaRESUMO
Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.
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Adenocarcinoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Pólipos Adenomatosos/genética , Éxons/genética , Mutação Puntual/genética , Neoplasias Gástricas/genética , Desequilíbrio Alélico/genética , Variações do Número de Cópias de DNA/genética , Exoma/genética , Feminino , Mucosa Gástrica/metabolismo , Ligação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Perda de Heterozigosidade , Masculino , Linhagem , Regiões Promotoras Genéticas/genéticaRESUMO
Genome-wide association studies (GWASs) have revealed increased breast cancer risk associated with multiple genetic variants at 5p12. Here, we report the fine mapping of this locus using data from 104,660 subjects from 50 case-control studies in the Breast Cancer Association Consortium (BCAC). With data for 3,365 genotyped and imputed SNPs across a 1 Mb region (positions 44,394,495-45,364,167; NCBI build 37), we found evidence for at least three independent signals: the strongest signal, consisting of a single SNP rs10941679, was associated with risk of estrogen-receptor-positive (ER+) breast cancer (per-g allele OR ER+ = 1.15; 95% CI 1.13-1.18; p = 8.35 × 10-30). After adjustment for rs10941679, we detected signal 2, consisting of 38 SNPs more strongly associated with ER-negative (ER-) breast cancer (lead SNP rs6864776: per-a allele OR ER- = 1.10; 95% CI 1.05-1.14; p conditional = 1.44 × 10-12), and a single signal 3 SNP (rs200229088: per-t allele OR ER+ = 1.12; 95% CI 1.09-1.15; p conditional = 1.12 × 10-05). Expression quantitative trait locus analysis in normal breast tissues and breast tumors showed that the g (risk) allele of rs10941679 was associated with increased expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in â¼10% of human breast cancers, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis.
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Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cromossomos Humanos Par 5/genética , Fator 10 de Crescimento de Fibroblastos/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores de Estrogênio/metabolismo , Alelos , Estudos de Casos e Controles , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Haplótipos/genética , Humanos , Regiões Promotoras Genéticas/genética , Locos de Características Quantitativas/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
The human immunodeficiency virus (HIV)-1 transactivator protein Tat is known to play a key role in HIV infection, integrally related to its role in the host cell nucleus/nucleolus. Here we show for the first time that Tat localisation can be modulated by specific methylation, whereby overexpression of active but not catalytically inactive PRMT6 methyltransferase specifically leads to exclusion of Tat from the nucleolus. An R52/53A mutated Tat derivative does not show this redistribution, implying that R52/53, within Tat's nuclear/nucleolar localisation signal, are the targets of PRMT6 activity. Analysis using fluorescence recovery after photobleaching indicate that Tat nucleolar accumulation is largely through binding to nucleolar components, with methylation of Tat by PRMT6 preventing this. To our knowledge, this is the first report of specific protein methylation inhibiting nucleolar retention.
Assuntos
Nucléolo Celular/metabolismo , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Células COS , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , HIV-1/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metilação , Microscopia Confocal , Mutação , Sinais de Localização Nuclear/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteína-Arginina N-Metiltransferases/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Proteína Vermelha FluorescenteRESUMO
Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3-4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target.
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Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Fator 1 de Elongação de Peptídeos/metabolismo , Transcrição Reversa/fisiologia , Replicação Viral/fisiologia , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , ImunoprecipitaçãoRESUMO
UNLABELLED: Previously, we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry assay. Mixtures of recombinant 6×His-RT and Nullbasic-FLAG-V5-6×His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced the levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less-dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic-treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic-treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge, this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability. IMPORTANCE: HIV-1 infection is treated by using combinations of antiretroviral drugs that target independent steps of virus replication. A newly described antiviral protein called Nullbasic can also inhibit a combination of different steps in virus replication (transcription, reverse transcription, and Rev-mediated viral mRNA transport), although the precise mechanism of action is unknown. This study shows that Nullbasic can inhibit reverse transcription by binding to the viral enzyme called reverse transcriptase, which results in accelerated uncoating of the viral core and instability of the viral apparatus called the reverse transcription complex (RTC). This unique antiviral activity may inform development of other RTC inhibitors, as well as providing a unique investigative tool for dissecting the RTC cellular composition.
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HIV-1/fisiologia , Transcrição Reversa , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Centrifugação , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Imunoprecipitação , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.
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Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Fator 1 de Elongação de Peptídeos/metabolismo , Linhagem Celular , Cromatografia Líquida , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Fator 1 de Elongação de Peptídeos/genética , RNA Interferente Pequeno , Espectrometria de Massas em Tandem , Transcrição GênicaRESUMO
BACKGROUND: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. RESULTS: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. CONCLUSIONS: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev's activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.
Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , HIV-1/fisiologia , Replicação Viral , Produtos do Gene rev do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , HIV-1/genética , Humanos , Imunoprecipitação , Espectrometria de Massas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mapeamento de Interação de Proteínas , Proteômica , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: Protein arginine methyltransferase 6 (PRMT6) can methylate the HIV-1 Tat, Rev and nucleocapsid proteins in a manner that diminishes each of their functions in in vitro assays, and increases the stability of Tat in human cells. In this study, we explored the relationship between PRMT6 and HIV-1 Tat by determining the domains in each protein required for interaction. METHODS: Through domain mapping and immunoprecipitation experiments, we determined that both the amino and carboxyl termini of PRMT6, and the activation domain within Tat are essential for interaction. Mutation of the basic domain of Tat did not affect the ability of PRMT6 to interact with Tat. RESULTS: We next used the A549 human alveolar adenocarcinoma cell line, which naturally expresses undetectable levels of PRMT6, as a model for testing the effects of PRMT6 on Tat stability, transactivation, and HIV-1 replication. As previously observed, steady state levels and the protein half-life of Tat were increased by the ectopic expression of PRMT6. However, no down regulation of Tat transactivation function was observed, even with over 300-fold molar excess of PRMT6 plasmid. We also observed no negative effect on HIV-1 infectivity when A549 producer cells overexpressed PRMT6. CONCLUSIONS: We show that PRMT6 requires the activation domain, but surprisingly not the basic domain, of Tat for protein interaction. This interaction between Tat and PRMT6 may impact upon pathogenic effects attributed to Tat during HIV-1 infection other than its function during transactivation.
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HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ativação Transcricional , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Células Epiteliais/virologia , Humanos , Imunoprecipitação , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de ProteínaRESUMO
The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/ß-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/ß nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin ß1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/ß nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.
Assuntos
Vírus da Dengue/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Ivermectina/farmacologia , Carioferinas/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Vírus da Dengue/fisiologia , HIV-1/fisiologia , Células HeLa , Humanos , Carioferinas/fisiologiaRESUMO
BACKGROUND: Schistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis. METHODS: We developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda. FINDINGS: The S. japonicum SHERLOCK assay achieved 93-100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples. INTERPRETATION: These schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases. FUNDING: Australian Infectious Diseases Research Centre seed grant (2022) and National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).
Assuntos
COVID-19 , Schistosoma japonicum , Esquistossomose , Humanos , Feminino , Animais , Camundongos , Sensibilidade e Especificidade , Austrália , Esquistossomose/diagnóstico , Teste para COVID-19RESUMO
BACKGROUND: Genome-wide association studies (GWAS) have identified > 200 loci associated with breast cancer risk. The majority of candidate causal variants are in non-coding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association, and identifying the phenotype it mediates, is a major challenge in the interpretation and translation of GWAS. RESULTS: Here, we show that pooled CRISPR screens are highly effective at identifying GWAS target genes and defining the cancer phenotypes they mediate. Following CRISPR mediated gene activation or suppression, we measure proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We perform 60 CRISPR screens and identify 20 genes predicted with high confidence to be GWAS targets that promote cancer by driving proliferation or modulating the DNA damage response in breast cells. We validate the regulation of a subset of these genes by breast cancer risk variants. CONCLUSIONS: We demonstrate that phenotypic CRISPR screens can accurately pinpoint the gene target of a risk locus. In addition to defining gene targets of risk loci associated with increased breast cancer risk, we provide a platform for identifying gene targets and phenotypes mediated by risk variants.
Assuntos
Estudo de Associação Genômica Ampla , Neoplasias , Animais , Camundongos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Predisposição Genética para Doença , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.
RESUMO
Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2'-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.