RESUMO
BACKGROUND: RNASEH1 (Ribonuclease H1) encodes an endonuclease that specifically degrades the RNA of RNA-DNA hybrids and acts in DNA replication and repair. Although there are many studies on RNASEH1, the research of RNASEH1 in cancers is still insufficient. Therefore, in order to clarify the physiological mechanism of RNASEH1 in tumor cells, we evaluated the role of RNASEH1 by combining The Cancer Genome Atlas (TCGA) pan-cancer data and Genotype-Tissue Expression (GTEx) normal tissue data. METHODS: RNASEH1 expression was analyzed by using RNAseq data from TCGA and the GTEx database. The Human Protein Atlas (HPA), GeneCards and STRING database were used to explore the protein information of RNASEH1. The prognostic value of RNASEH1 was analyzed by using the clinical survival data from TCGA. Differential analysis of RNASEH1 in different cancers was performed by using R package "DESeq2", and enrichment analysis of RNASEH1 was conducted by using R package "clusterProfiler". We downloaded the immune cell infiltration score of TCGA samples from published articles and online databases, and the correlation analysis between immune cell infiltration levels and RNASEH1 expression was performed. Not only that, we further evaluated the association of RNASEH1 with immune activating genes, immunosuppressive genes, chemokines and chemokine receptors. At the end of the article, the differential expression of RNASEH1 in pan-cancer was validated by using GSE54129, GSE40595, GSE90627, GSE106937, GSE145976 and GSE18672, and qRT-PCR was also performed for verification. FINDINGS: RNASEH1 was significantly overexpressed in 19 cancers and the overexpression was closely correlated with poor prognosis. Moreover, the expression of RNASEH1 was significantly correlated with the regulation of the tumor microenvironment. In addition, RNASEH1 expression was closely associated with immune cell infiltration, immune checkpoints, immune activators, immunosuppressive factors, chemokines and chemokine receptors. Finally, RNASEH1 also was closely associated with DNA-related physiological activities and mitochondrial-related physiological activities. INTERPRETATION: Our studying suggests that RNASEH1 is a potential cancer biomarker. And RNASEH1 may be able to regulate the tumor microenvironment by regulating the relevant physiological activities of mitochondrial and thereby regulating the occurrence and development of tumors. Thus, it could be used to develop new-targeted drugs of tumor therapy.
Assuntos
Neoplasias , Ribonuclease H , Microambiente Tumoral , Humanos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/mortalidade , Neoplasias/patologia , Ribonuclease H/análise , Ribonuclease H/genética , Expressão Gênica , Mutação , Metilação de DNA , Prognóstico , Análise de Sobrevida , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologiaRESUMO
OBJECTIVES: RNASEH1 gene has recently been associated with type 1 diabetes (T1D) in Colombia. The purpose of this study was to fine mapping the putative functional variant in RNASEH1 and testing its interaction with HLA tagSNPs. METHODS: Two-hundred nuclear families with T1D were included in this study. Probands were tested for GAD65 and IA-2 autoantibodies. Genotyping was performed using 20 coding tagSNPs uncovered through Sanger sequencing (N = 96), in addition to 23 tagSNPs chosen from 1000genomes to cover the extent of the gene region. Also, 45 tagSNPs for classic HLA alleles associated with T1D were also genotyped. The transmission disequilibrium test (TDT) was used to test for association and a multiple testing correction was made using permutation. Interaction between RNASEH1 variants and HLA was evaluated by means of the M-TDT test. RESULTS: We identified 20 variants (15 were novel) in the 96 patients sequenced. None of these variants were in linkage disequilibrium. In total, 43 RNASEH1 variants were genotyped in the 200 families. Association between T1D and rs7607888 was identified (P = .002). Haplotype analysis involving rs7607888 variant revealed even stronger association with T1D (most significative P = .0003). HLA tagSNPs displayed stronger associations (OR = 6.39, 95% CI = 4.33-9.44, P-value = 9.74E-28). Finally, we found several statistically significant interactions of HLA variants with rs7607888 (P-value ranged from 8.77E-04 to 5.33E-12). CONCLUSION: Our results verify the association of rs7607888 in RNASEH1 gene with T1D. It is also shown in the interaction between RNASEH1 and HLA for conveying risk to T1D in Northwest Colombia. Work is underway aiming to identify the actual classic HLA alleles associated with the tagSNPs tested here.
Assuntos
Diabetes Mellitus Tipo 1/genética , Antígenos de Histocompatibilidade Classe II/genética , Polimorfismo de Nucleotídeo Único/genética , Ribonuclease H/genética , Autoanticorpos/sangue , Criança , Pré-Escolar , Colômbia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Feminino , Haplótipos , Humanos , MasculinoRESUMO
BACKGROUND: In a previous work, we found linkage and association of type 1 diabetes (T1D) to a 12 known gene region at chromosome 2p25 in Colombian families. Here, we present further work on this candidate region. MATERIALS AND METHODS: Seventeen SNPs located on the 12 candidate genes, in 100 familial trios set, were tested by ARMS-tetraprimer-PCR or PCR-RFLP. Five extra SNPs in the vicinity of rs10186193 were typed. A replica phase included 97 novel familial trios, in whom diabetes-related auto-antibodies (AABs) were tested in sera of the patients. In addition to transmission disequilibrium tests, haplotype analyses were carried out using the unphased software. RESULTS: SNP rs10186193 (at RNASEH1 gene) showed association with T1D (P = 0.005). The additional five SNPs revealed that rs7607888 (P = 2.03 × 10-7), rs55981318 (P = 0.018), and rs1136545 (P = 1.93 × 10-9) were also associated with T1D. Haplotype analysis showed association for rs55981318-rs10186193 (P = 0.0005), rs7563960-rs7607888 (P = 0.0007), rs7607888-rs1136545 (P = 9.21 × 10-10), and rs1136545-rs11538545 (P = 6.67 × 10-8). In contrast, the new set of 97 familial trios tested for SNPs rs55981318, rs10186193, and rs7607888 did not support the previous finding; however, by combining the sample (197 trios), evidence of association of T1D with rs55981318 and rs7607888 was conclusive. In addition, a two-loci haplotype analysis of the combined sample showed significant association of RNASEH1 with T1D (P = 3.1 × 10-5). CONCLUSION: In conclusion, our analyses suggest that RNASEH1 gene variants associate with susceptibility/protection to T1D in Colombia.
Assuntos
Diabetes Mellitus Tipo 1/genética , Polimorfismo de Nucleotídeo Único , Ribonuclease H/genética , Adulto , Criança , Colômbia/epidemiologia , Diabetes Mellitus Tipo 1/epidemiologia , Família , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Polimorfismo de Fragmento de RestriçãoRESUMO
Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%-73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.
Assuntos
Caulimoviridae/classificação , Caulimoviridae/isolamento & purificação , Elaeocarpaceae/virologia , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Caulimoviridae/genética , Análise por Conglomerados , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Proteínas do Movimento Viral em Plantas/genética , DNA Polimerase Dirigida por RNA/genética , Ribonuclease H/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Integração ViralRESUMO
Two new sesquiterpenoid tropolone glycosides, liriosmasides A (1) and B (2), along with two known compounds, secoxyloganin and oplopanpheside C, were isolated from a methanol extract of the roots of Liriosma ovata. The structures of 1 and 2 were elucidated by spectroscopic methods including 1D and 2D NMR and by high-resolution mass spectrometry involving an ultra-high-performance liquid chromatography-quadrupole-orbital ion trap mass spectrometric (UHPLC-Q-Orbitrap MS) method. Compound 1 showed weak inhibitory activity against HIV RNase H.
Assuntos
Glicosídeos/isolamento & purificação , Olacaceae/química , Sesquiterpenos/isolamento & purificação , Tropolona/análogos & derivados , Tropolona/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Glicosídeos/química , Glicosídeos/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peru , Raízes de Plantas/química , Ribonuclease H/antagonistas & inibidores , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Tropolona/química , Tropolona/farmacologiaRESUMO
High telomerase activity is always associated with actively dividing cells, however the detection of this activity in dividing Leishmania and Trypanosoma cruzi cells has always been disappointingly low. Recently, we have found that Leishmania major telomerase activity can be activated by heat, which combined with dilutions of the nuclear extracts produced an increase in activity comparable to cancer cells. Here we examined whether T. cruzi telomerase shares the same physicochemical properties of primer specificity and overall features of the L. major. Our studies revealed that no telomerase inhibitory factors were present in the nuclear lysates of T. cruzi however the enzyme was activated by heat and was very resilient to heat denaturation. We also showed the extension primer specificity, susceptibility to RNase-A and RNase-H digestion, and the effect of telomerase inhibitors.
Assuntos
Telomerase/metabolismo , Trypanosoma cruzi/enzimologia , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Temperatura Alta , Desnaturação Proteica/efeitos da radiação , Estabilidade Proteica , Ribonuclease H/metabolismo , Ribonuclease Pancreático/metabolismo , Especificidade por Substrato , Telomerase/antagonistas & inibidores , Telomerase/químicaRESUMO
A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg(2+)-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg(2+)-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar K(m)s for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Avaliação Pré-Clínica de Medicamentos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/metabolismo , Magnésio/metabolismo , Modelos Moleculares , RNA de Transferência/genética , Ribonuclease H/química , Ribonuclease H/genética , Análise de Sequência de RNARESUMO
BACKGROUND: Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. METHODS AND FINDINGS: We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. CONCLUSIONS: This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.
Assuntos
Sequência Conservada , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/genética , Mutação , Inibidores da Transcriptase Reversa/uso terapêutico , Ribonuclease H/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Infecções por HIV/enzimologia , Transcriptase Reversa do HIV/química , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
VIPER was initially characterized as a 2326bp LTR-like retroelement associated to SIRE, a short interspersed repetitive element specific of Trypanosoma cruzi. It carried a single ORF that coded for a putative reverse transcriptase-RNAse H protein, suggesting that it could be a truncated copy of a longer retroelement. Herein we report the identification and characterization of a complete 4480bp long VIPER in the T. cruzi genome. The complete VIPER harbored three non-overlapped domains encoding for a GAG-like, a tyrosine recombinase and a reverse transcriptase-RNAse H proteins. VIPER elements were also found in the genomes of Trypanosoma brucei and Trypanosoma vivax, but not in Leishmania sp. On the basis of its reverse transcriptase phylogeny, VIPER was classified as an LTR retroelement. However, VIPER was structurally related to the tyrosine recombinase encoding retroelements, DIRS and Ngaro. Phylogenetic analysis showed that VIPER's tyrosine recombinase grouped with the transposases RCI1 of Escherichia coli and Ye24 and Ye72 of Haemophilus influenzae within a major branch of prokaryotic recombinases. Taken together, VIPER's structure, the nature of its tyrosine recombinase, the unique features of its reverse transcriptase catalytic consensus motif and the fact that it was found in Trypanosomes, an early branching eukaryote, suggest that VIPER may be the closest relative of the founder element of the tyrosine recombinase encoding retrotransposons known up to date. Our analysis revealed that tyrosine recombinase-encoding retroelements were originated as early in evolution as non-LTR retroelements and suggests that VIPER, Ngaro and DIRS elements may constitute a third group of retrotransposons, distinct from both LTR and non-LTR retroelements.
Assuntos
Genoma de Protozoário , Recombinases/genética , Retroelementos/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , DNA Nucleotidiltransferases/genética , Produtos do Gene gag/genética , Haemophilus influenzae/genética , Leishmania/genética , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , DNA Polimerase Dirigida por RNA/genética , Ribonuclease H/genética , Homologia de Sequência , Trypanosoma brucei brucei/genética , Trypanosoma vivax/genéticaRESUMO
The C-terminus of the HIV-1 reverse transcriptase heterodimer was reconstructed into a single polypeptide. The construct encodes the p51 thumb (T) and connection (C) subdomains joined through a linker region to the p66 connection (C) and RNase H (R) domain. The TCCR protein was purified from insoluble fractions of Escherichia coli lysates. The TCCR construct maintains Mn(2+)-dependent RNase H activity and specifically cleaves the substrate mimicking the tRNA removal required for second-strand transfer reactions.