ABSTRACT
BACKGROUND: SINE-VNTR-Alu (SVA) retrotransposons move from one genomic location to another in a 'copy-and-paste' manner. They continue to move actively and cause monogenic diseases through various mechanisms. Currently, disease-causing SVA retrotransposons are classified into human-specific young SVA_E or SVA_F subfamilies. In this study, we identified an evolutionarily old SVA_D retrotransposon as a novel cause of occipital horn syndrome (OHS). OHS is an X-linked, copper metabolism disorder caused by dysfunction of the copper transporter, ATP7A. METHODS: We investigated a 16-year-old boy with OHS whose pathogenic variant could not be detected via routine molecular genetic analyses. RESULTS: A 2.8 kb insertion was detected deep within the intron of the patient's ATP7A gene. This insertion caused aberrant mRNA splicing activated by a new donor splice site located within it. Long-read circular consensus sequencing enabled us to accurately read the entire insertion sequence, which contained highly repetitive and GC-rich segments. Consequently, the insertion was identified as an SVA_D retrotransposon. Antisense oligonucleotides (AOs) targeting the new splice site restored the expression of normal transcripts and functional ATP7A proteins. AO treatment alleviated excessive accumulation of copper in patient fibroblasts in a dose-dependent manner. Pedigree analysis revealed that the retrotransposon had moved into the OHS-causing position two generations ago. CONCLUSION: This is the first report of a human monogenic disease caused by the SVA_D retrotransposon. The fact that the evolutionarily old SVA_D is still actively transposed, leading to increased copy numbers may make a notable impact on rare genetic disease research.
ABSTRACT
Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon-intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.
Subject(s)
Oligonucleotides, Antisense , RNA Splicing , Female , Humans , Adolescent , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Mutation , Exons/genetics , Carrier Proteins/geneticsABSTRACT
BACKGROUND: Currently, only limited knowledge is available regarding the phenotypic association between fibroblast growth factor receptor 3 (FGFR3) alterations and the tumor microenvironment (TME) in bladder cancer (BLCA). METHODS: A multi-omics analysis on 389 BLCA and 35 adjacent normal tissues from a cohort of OMPU-NCC Consortium Japan was retrospectively performed by integrating the whole-exome and RNA-sequence dataset and clinicopathological record. A median follow-up duration of all BLCA cohort was 31 months. RESULTS: FGFR3 alterations (aFGFR3), including recurrent mutations and fusions, accounted for 44% of non-muscle invasive bladder cancer (NMIBC) and 15% of muscle-invasive bladder cancer (MIBC). Within MIBC, the consensus subtypes LumP was significantly more prevalent in aFGFR3, whereas the Ba/Sq subtype exhibited similarity between intact FGFR3 (iFGFR3) and aFGFR3 cases. We revealed that basal markers were significantly increased in MIBC/aFGFR3 compared to MIBC/iFGFR3. Transcriptome analysis highlighted TIM3 as the most upregulated immune-related gene in iFGFR3, with differential immune cell compositions observed between iFGFR3 and aFGFR3. Using EcoTyper, TME heterogeneity was discerned even within aFGFR cases, suggesting potential variations in the response to checkpoint inhibitors (CPIs). Among 72 patients treated with CPIs, the objective response rate (ORR) was comparable between iFGFR3 and aFGFR3 (20% vs 31%; p = 0.467). Strikingly, a significantly higher ORR was noted in LumP/aFGFR3 compared to LumP/iFGFR3 (50% vs 5%; p = 0.022). This trend was validated using data from the IMvigor210 trial. Additionally, several immune-related genes, including IDO1, CCL24, IL1RL1, LGALS4, and NCAM (CD56) were upregulated in LumP/iFGFR3 compared to LumP/aFGFR3 cases. CONCLUSIONS: Differential pathways influenced by aFGFR3 were observed between NMIBC and MIBC, highlighting the upregulation of both luminal and basal markers in MIBC/aFGFR3. Heterogeneous TME was identified within MIBC/aFGFR3, leading to differential outcomes for CPIs. Specifically, a favorable ORR in LumP/aFGFR3 and a poor ORR in LumP/iFGFR3 were observed. We propose TIM3 as a potential target for iFGFR3 (ORR: 20%) and several immune checkpoint genes, including IDO1 and CCL24, for LumP/iFGFR3 (ORR: 5%), indicating promising avenues for precision immunotherapy for BLCA.
Subject(s)
Immune Checkpoint Inhibitors , Urinary Bladder Neoplasms , Humans , Prognosis , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Biomarkers, Tumor/genetics , Retrospective Studies , Receptor, Fibroblast Growth Factor, Type 3/genetics , Tumor Microenvironment , Hepatitis A Virus Cellular Receptor 2 , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
BACKGROUND: Deep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants. RESULTS: PDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors. CONCLUSION: Incorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .
Subject(s)
RNA Splicing , Humans , Introns , Virulence , MutationABSTRACT
Emerging evidence suggests that the presence of tertiary lymphoid structures (TLS) and neutrophil-lymphocyte ratio (NLR) in peripheral blood is associated with the treatment response to checkpoint inhibitors (CPIs), whereas there is limited knowledge regarding whether these factors reciprocally impact the treatment outcomes of CPIs in metastatic urothelial carcinoma (mUC). Herein, we investigated treatment outcomes of platinum-refractory mUC patients (50 cases with whole-exome and transcriptome sequencing) treated with pembrolizumab. The pathological review identified 24% of cases of TLS in the specimens. There was no significant difference in the NLR between the TLS- and TLS+ groups (p = 0.153). In the lower NLR group, both overall survival and progression-free survival were significantly longer in patients with TLS than in those without TLS, whereas the favorable outcomes associated with TLS were not observed in patients in the higher NLR group. We explored transcriptomic differences in UC with TLS. The TLS was comparably observed between luminal (20%) and basal (25%) tumor subtypes (p = 0.736). Exploring putative immune-checkpoint genes revealed that ICOSLG (B7-H2) was significantly increased in tumors with lower NLR. KRT expression levels exhibited higher basal cell markers (KRT5 and KRT17) in the higher NLR group and lower differentiated cell markers (KRT8 and KRT18) in patients with TLS. In conclusion, the improved outcomes of pembrolizumab treatment in mUC are restricted to patients with lower NLR. Our findings begin to elucidate a distinct molecular pattern for the presence of TLS according to the NLR in peripheral blood.
Subject(s)
Carcinoma, Transitional Cell , Tertiary Lymphoid Structures , Urinary Bladder Neoplasms , Humans , Neutrophils , Lymphocytes , Prognosis , Retrospective StudiesABSTRACT
Chronic infection with Kaposi's sarcoma-associated herpes virus (KSHV) in B lymphocytes causes primary effusion lymphoma (PEL), the most aggressive form of KSHV-related cancer, which is resistant to conventional chemotherapy. In this study, we report that the BCBL-1 KSHV+ PEL cell line does not harbor oncogenic mutations responsible for its aggressive malignancy. Assuming that KSHV viral oncogenes play crucial roles in PEL proliferation, we examined the effect of cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on KSHV viral gene expression and KSHV+ PEL proliferation. We found that FIT-039 treatment impaired the proliferation of KSHV+ PEL cells and the expression of KSHV viral genes in vitro. The effects of FIT-039 treatment on PEL cells were further evaluated in the PEL xenograft model that retains a more physiological environment for the growth of PEL growth and KSHV propagation, and we confirmed that FIT-039 administration drastically inhibited PEL growth in vivo. Our current study indicates that FIT-039 is a potential new anticancer drug targeting KSHV for PEL patients.
Subject(s)
Herpesvirus 8, Human , Lymphoma, Primary Effusion , Neoplasms , Sarcoma, Kaposi , Humans , Sarcoma, Kaposi/drug therapy , Lymphoma, Primary Effusion/pathology , Cyclin-Dependent Kinase 9/metabolismABSTRACT
Neoantigen production is a determinant of cancer immunotherapy. However, the expansion of neoantigen abundance for cancer therapeutics is technically challenging. Here, we report that the synthetic compound RECTAS can induce the production of splice-neoantigens that could be used to boost antitumor immune responses. RECTAS suppressed tumor growth in a CD8+ T cell- and tumor major histocompatibility complex class I-dependent manner and enhanced immune checkpoint blockade efficacy. Subsequent transcriptome analysis and validation for immunogenicity identified six splice-neoantigen candidates whose expression was induced by RECTAS treatment. Vaccination of the identified neoepitopes elicited T cell responses capable of killing cancer cells in vitro, in addition to suppression of tumor growth in vivo upon sensitization with RECTAS. Collectively, these results provide support for the further development of splice variant-inducing treatments for cancer immunotherapy.
Subject(s)
Colorectal Neoplasms , Immunotherapy , Humans , Mutation , CD8-Positive T-Lymphocytes , Gene Expression Profiling , Colorectal Neoplasms/therapyABSTRACT
Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.
Subject(s)
Alternative Splicing/genetics , Dysautonomia, Familial/genetics , Mutation , Transcriptional Elongation Factors/genetics , Alternative Splicing/drug effects , Animals , Cells, Cultured , Disease Models, Animal , Dysautonomia, Familial/drug therapy , Dysautonomia, Familial/metabolism , Enhancer Elements, Genetic/genetics , Exons/genetics , HeLa Cells , Humans , Introns/genetics , Mice, Transgenic , Molecular Structure , Phosphoproteins/metabolism , Protein Binding/drug effects , RNA Splice Sites/genetics , Serine-Arginine Splicing Factors/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcriptional Elongation Factors/metabolismABSTRACT
Dysregulation of alternative splicing is a feature of cancer, both in aetiology and progression. It occurs because of mutations in splice sites or sites that regulate splicing, or because of the altered expression and activity of splice factors and of splice factor kinases that regulate splice factor activity. Recently the CDC2-like kinases (CLKs) have attracted attention due to their increasing involvement in cancer. We measured the effect of the CLK inhibitor, the benzothiazole TG003, on two prostate cancer cell lines. TG003 reduced cell proliferation and increased apoptosis in PC3 and DU145 cells. Conversely, the overexpression of CLK1 in PC3 cells prevented TG003 from reducing cell proliferation. TG003 slowed scratch closure and reduced cell migration and invasion in a transwell assay. TG003 decisively inhibited the growth of a PC3 cell line xenograft in nude mice. We performed a transcriptomic analysis of cells treated with TG003. We report widespread and consistent changes in alternative splicing of cancer-associated genes including CENPE, ESCO2, CKAP2, MELK, ASPH and CD164 in both HeLa and PC3 cells. Together these findings suggest that targeting CLKs will provide novel therapeutic opportunities in prostate cancer.
Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Molecular Targeted Therapy , Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Alternative Splicing/genetics , Animals , Apoptosis/drug effects , Benzothiazoles/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Male , Mice, Nude , Neoplasm Invasiveness , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , RNA-Seq , Thiazoles/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that compromise its chloride channel activity. Here, we present a therapeutic strategy to ameliorate RNA splicing deficiency of CFTR with a small molecule. The 3,849 + 10 kb C>T is the most common splicing mutation in CF, creating a pseudo exon with premature stop codon. We reveal that the 3,849 + 10 kb C>T-induced CFTR pseudo exon is regulated by phosphorylation of serine/arginine-rich splicing factors, and their functional inhibition by a CDC-like kinase inhibitor restores normal splicing of CFTR. Subsequent screening of our focused chemical library identified CaNDY as a rectifier of the aberrant splicing. CaNDY treatment restored normal splicing of CFTR with the 3,849 + 10 kb C>T in CF patient cells and functional CFTR protein expression in the CF model cells. Our findings open the door for mechanism-based personalized medicine for pseudo-exon-type genetic diseases.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Exons/genetics , Precision Medicine , Humans , Phosphorylation/drug effectsABSTRACT
Alternative RNA splicing is an important focus in molecular and clinical oncology. We report here that SRSF3 regulates alternative RNA splicing of interleukin enhancer binding factor 3 (ILF3) and production of this double-strand RNA-binding protein. An increased coexpression of ILF3 isoforms and SRSF3 was found in various types of cancers. ILF3 isoform-1 and isoform-2 promote cell proliferation and transformation. Tumor cells with reduced SRSF3 expression produce aberrant isoform-5 and -7 of ILF3. By binding to RNA sequence motifs, SRSF3 regulates the production of various ILF3 isoforms by exclusion/inclusion of ILF3 exon 18 or by selection of an alternative 3' splice site within exon 18. ILF3 isoform-5 and isoform-7 suppress tumor cell proliferation and the isoform-7 induces cell apoptosis. Our data indicate that ILF3 isoform-1 and isoform-2 are two critical factors for cell proliferation and transformation. The increased SRSF3 expression in cancer cells plays an important role in maintaining the steady status of ILF3 isoform-1 and isoform-2.
Subject(s)
Alternative Splicing , Cell Proliferation/genetics , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Nuclear Factor 90 Proteins/genetics , Serine-Arginine Splicing Factors/genetics , Animals , Apoptosis/genetics , Binding Sites , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Exons , HeLa Cells , Humans , Introns , Mice , NIH 3T3 Cells , Nuclear Factor 90 Proteins/metabolism , Osteoblasts/metabolism , Osteoblasts/pathology , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Serine-Arginine Splicing Factors/metabolismABSTRACT
X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic IKBKG (also known as NEMO) mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose 3 boys with EDA-ID died from ID. We identify the same private variant in an intron of IKBKG, IVS4+866 C>T, which was inherited from and occurred de novo in the European mother and Japanese mother, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudoexon (PE) generating a frameshift. Its leakiness accounts for NF-κB activation being impaired but not abolished in the boys' cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in induced pluripotent stem cell-derived (iPSC-derived) macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the PE, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.
Subject(s)
Ectodermal Dysplasia , Frameshift Mutation , I-kappa B Kinase , Incontinentia Pigmenti , Introns , Ectodermal Dysplasia/genetics , Ectodermal Dysplasia/metabolism , Ectodermal Dysplasia/pathology , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , I-kappa B Kinase/deficiency , I-kappa B Kinase/metabolism , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolism , Immunologic Deficiency Syndromes/pathology , Incontinentia Pigmenti/genetics , Incontinentia Pigmenti/metabolism , Incontinentia Pigmenti/pathology , Macrophages/metabolism , Macrophages/pathology , MaleABSTRACT
Purpose: Cervical cancer is one of the leading causes of cancer-related deaths among women worldwide. The purpose of this study is to assess the therapeutic effect of the newly developed cyclin-dependent kinase 9 (CDK9) inhibitor FIT-039 on cervical neoplasia induced by human papillomavirus (HPV) infection.Experimental Design: We examined FIT-039 for its effect on HPV gene expression in HPV+ cervical cancer cells. Primary keratinocytes monolayer and organotypic raft culture models were used to evaluate HPV viral replication and cervical intraepithelial neoplasia (CIN) phenotypes. Preclinical pharmacokinetics and toxicity tests for FIT-039 were also conducted. Finally, the anti-HPV effect of FIT-039 was further examined in vivo, using HPV+ cervical cancer xenografts.Results: FIT-039 inhibits HPV replication and expression of E6 and E7 viral oncogenes, restoring tumor suppressors p53 and pRb in HPV+ cervical cancer cells. The therapeutic effect of FIT-039 was demonstrated in CIN model of an organotypic raft culture, where FIT-039 suppressed HPV18-induced dysplasia/hyperproliferation with reduction in viral load. FIT-039 also repressed growth of HPV16+, but not HPV- cervical cancer xenografts without any significant adverse effects. Safety and pharmacokinetics of FIT-039 were confirmed for systemic and topical routes.Conclusions: The CDK9 inhibitor FIT-039 showed potent anti-HPV activity without significant toxicity in preclinical studies. Thus, FIT-039 is expected to be a novel therapeutic for CIN to prevent cervical cancer. Clin Cancer Res; 24(18); 4518-28. ©2018 AACR.
Subject(s)
Cyclin-Dependent Kinase 9/antagonists & inhibitors , Papillomavirus Infections/drug therapy , Pyridines/pharmacology , Uterine Cervical Dysplasia/drug therapy , Animals , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9/genetics , Female , Gene Expression Regulation, Viral/drug effects , Heterografts , Human papillomavirus 16/genetics , Human papillomavirus 16/pathogenicity , Humans , Keratinocytes/drug effects , Keratinocytes/virology , Mice , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , Primary Cell Culture , Repressor Proteins/genetics , Tumor Suppressor Protein p53/genetics , Virus Replication/drug effects , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/pathology , Uterine Cervical Dysplasia/virologyABSTRACT
The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors.IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter.
Subject(s)
DNA, Viral/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Human papillomavirus 18/genetics , Promoter Regions, Genetic , Transcription, Genetic , Virus Replication/genetics , Aphidicolin/pharmacology , DNA Replication , Gene Expression Regulation, Viral , Genes, Viral , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Host-Pathogen Interactions , Human papillomavirus 18/metabolism , Humans , Keratinocytes/virology , RNA Splicing , Replication Origin/geneticsABSTRACT
Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus with an estimated infection in â¼180 million people worldwide, and its chronic infection leads to development of cirrhosis and hepatocellular carcinoma. Although recent development of direct acting antiviral (DAA) compounds improved anti-HCV regimens, alternative therapeutic compounds are still demanded due to an expected emergence of escape mutants for those DAAs. In order to identify novel anti-HCV agents, we conducted chemical library screening for 2086 compounds using HCV Rep-Feo reporter replicon in Huh7 hepatoma cells. Our screening identified retinoid derivative Tp80, which inhibits replication of HCV Rep-Feo (genotype 1b) and JFH1 HCV (genotype 2a) with 0.62 µM and 1.0 µM, respectively, of 50% effective concentration (EC50 ), at which cytotoxicity is not evident for host hepatocytes. Subsequent transcriptome profiling revealed Tp80 exhibits anti-HCV activity through restoration of gastrointestinal glutathione peroxidase (GI-GPx), suppression of which is responsible for HCV-induced oxidative stress to facilitate HCV replication. Furthermore, comparison of Tp80 with other retinoid derivatives revealed Tp80 shows best potency in both GI-GPx restoration and anti-HCV activity among compounds we examined. In conclusion, our current study provides Tp80 as a promising candidate of anti-HCV compound, suppressing host cellular oxidative stress through a restoration of GI-GPx.
Subject(s)
Antiviral Agents/pharmacology , Glutathione Peroxidase/genetics , Hepacivirus/drug effects , Retinoids/pharmacology , Virus Replication/drug effects , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/virology , Cell Line , Drug Discovery , Gene Expression Profiling , Genotype , Glutathione Peroxidase/metabolism , Hepacivirus/genetics , Hepatocytes/drug effects , Hepatocytes/virology , High-Throughput Screening Assays , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/virology , Oxidative Stress , Retinoids/chemistry , Small Molecule LibrariesABSTRACT
UNLABELLED: Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCE: Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.
Subject(s)
Alternative Splicing , Gene Expression Regulation, Viral , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Host-Pathogen Interactions , Human papillomavirus 18/physiology , Serine-Arginine Splicing Factors/metabolism , Binding Sites , Gene Knockdown Techniques , Heterogeneous Nuclear Ribonucleoprotein A1 , Human papillomavirus 18/genetics , Humans , Point Mutation , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Viral/genetics , RNA, Viral/metabolismABSTRACT
Alternative RNA splicing is an essential process to yield proteomic diversity in eukaryotic cells, and aberrant splicing is often associated with numerous human diseases and cancers. We recently described serine/arginine-rich splicing factor 3 (SRSF3 or SRp20) being a proto-oncogene. However, the SRSF3-regulated splicing events responsible for its oncogenic activities remain largely unknown. By global profiling of the SRSF3-regulated splicing events in human osteosarcoma U2OS cells, we found that SRSF3 regulates the expression of 60 genes including ERRFI1, ANXA1 and TGFB2, and 182 splicing events in 164 genes, including EP300, PUS3, CLINT1, PKP4, KIF23, CHK1, SMC2, CKLF, MAP4, MBNL1, MELK, DDX5, PABPC1, MAP4K4, Sp1 and SRSF1, which are primarily associated with cell proliferation or cell cycle. Two SRSF3-binding motifs, CCAGC(G)C and A(G)CAGCA, are enriched to the alternative exons. An SRSF3-binding site in the EP300 exon 14 is essential for exon 14 inclusion. We found that the expression of SRSF1 and SRSF3 are mutually dependent and coexpressed in normal and tumor tissues/cells. SRSF3 also significantly regulates the expression of at least 20 miRNAs, including a subset of oncogenic or tumor suppressive miRNAs. These data indicate that SRSF3 affects a global change of gene expression to maintain cell homeostasis.
Subject(s)
MicroRNAs/biosynthesis , Osteosarcoma/genetics , RNA Splicing/genetics , RNA-Binding Proteins/biosynthesis , Binding Sites , Cell Line, Tumor , Exons , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , Osteosarcoma/pathology , Proteomics , Proto-Oncogene Mas , RNA-Binding Proteins/genetics , Serine-Arginine Splicing FactorsABSTRACT
Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression.
Subject(s)
Argonaute Proteins/metabolism , Cell Nucleus/metabolism , Gene Expression Regulation , Papillomavirus Infections/metabolism , Cell Line, Tumor , Cervix Uteri/metabolism , Cervix Uteri/virology , Female , Gene Silencing , HEK293 Cells , HeLa Cells , Human papillomavirus 16 , Human papillomavirus 18 , Humans , Keratinocytes/cytology , Larynx/metabolism , Larynx/virology , RNA, Small Interfering/metabolism , Skin/metabolism , Skin/virology , Subcellular Fractions , Tissue Distribution , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/virologyABSTRACT
One mechanism of resistance of the melanoma-associated BRAF kinase to its small molecule inhibitor vemurafenib is by point mutations in its intron 8 resulting in exons 4-8 skipping. In this report, we carried out in vitro BRAF RNA splicing assays and lariat RT-PCR to map the intron 8 branch points in wild-type and BRAF mutants. We identify multiple branch points (BP) in intron 8 of both wild-type (wt) and vemurafenib-resistant BRAF RNA. In wt BRAF, BPs are located at -29A, -28A and -26A, whereas in a vemurafenib-resistant BRAF splicing mutant, BPs map to -22A, -18A and -15A, proximal to the intron 8 3' splice site. This finding of a distal-to-proximal shift of the branch point sequence in BRAF splicing in response to point-mutations in intron 8 provides insight into the regulation of BRAF alternative splicing upon vemurafenib resistance.