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1.
Protein & Cell ; (12): 540-552, 2018.
Artigo em Inglês | WPRIM | ID: wpr-758003

RESUMO

Natural antisense transcripts (NAT) and alternative polyadenylation (APA) of messenger RNA (mRNA) are important contributors of transcriptome complexity, each playing a critical role in multiple biological processes. However, whether they have crosstalk and function collaboratively is unclear. We discovered that APA enriched in human sense-antisense (S-AS) gene pairs, and finally focused on RNASEH2C-KAT5 S-AS pair for further study. In cis but not in trans over-expression of the antisense KAT5 gene promoted the usage of distal polyA (pA) site in sense gene RNASEH2C, which generated longer 3' untranslated region (3'UTR) and produced less protein, accompanying with slowed cell growth. Mechanistically, elevated Pol II occupancy coupled with SRSF3 could explain the higher usage of distal pA site. Finally, NAT-mediated downregulation of sense gene's protein level in RNASEH2C-KAT5 pair was specific for human rather than mouse, which lacks the distal pA site of RNASEH2C. We provided the first evidence to support that certain gene affected phenotype may not by the protein of its own, but by affecting the expression of its overlapped gene through APA, implying an unexpected view for understanding the link between genotype and phenotype.


Assuntos
Humanos , Proliferação de Células , Genética , Evolução Molecular , Regulação da Expressão Gênica , Genética , Células HEK293 , Poliadenilação , Genética , RNA Antissenso , Genética , RNA Mensageiro , Genética , Ribonuclease H , Genética , Fatores de Processamento de Serina-Arginina , Metabolismo , Transcrição Gênica , Regulação para Cima , Genética
2.
The Korean Journal of Parasitology ; : 451-455, 2017.
Artigo em Inglês | WPRIM | ID: wpr-69358

RESUMO

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3′-O-P bond of RNA in a DNA-RNA duplex, producing 3′-hydroxyl and 5′-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.


Assuntos
Aminoácidos , Domínio Catalítico , DNA Complementar , Echinostoma , Endorribonucleases , Escherichia coli , Intestino Delgado , Oligonucleotídeos Antissenso , Parasitos , Ribonuclease H , Ribonucleases , RNA , Trematódeos
3.
Acta Pharmaceutica Sinica ; (12): 860-865, 2013.
Artigo em Chinês | WPRIM | ID: wpr-259539

RESUMO

This study is to investigate the mechanism and action characteristics of 6-chloro-3-methyl-4-(2-methyoxycarbonylthiophene-3-sulfonyl)-3, 4-dihydroquinoxa-lin-2-(1 H)-one (XU07011) against HIV-1 replication. XU07011 anti-HIV activity was tested by using VSVG/HIV pseudotype viral system and confirmed by HIV-1 live viruses' infectious assay. Time of addition was used to test HIV-1 reverse transcription process. RNA-dependent DNA polymerase activity and RNase H activity were tested by using enzyme linked immunoabsorbent assay and fluorescence method. Wild type and nine NNRTIs-resistant reverse transcriptase enzymatic models and cell-based pharmacological models were used to evaluate XU07011 bio-characteristics. The results showed that XU07011 inhibited HIV-1 replication with IC50 of (0.057 +/- 0.01) micromol x L(-1) which was comparable to nevirapine [IC50: (0.046 +/- 0.01) micromol x L(-1)]. Mechanism study data indicated that XU07011 blocked HIV-1 reverse transcription process through acting on reverse transcriptase RNA-dependent DNA polymerase with IC 50 of (1.1 +/- 0.3) micromol x L(-1). The compound showed no effect on RNase H activity. XU07011 exhibited better activities comparing with nevirapine on K103N mutated NNRTIs-resistant HIV-1 strains. This study could provide a theoretical basis for novel anti-HIV reagents development.


Assuntos
Humanos , Fármacos Anti-HIV , Química , Farmacologia , Farmacorresistência Viral , Células HEK293 , HIV-1 , Fisiologia , Concentração Inibidora 50 , Estrutura Molecular , Nevirapina , Farmacologia , Quinoxalinas , Farmacologia , DNA Polimerase Dirigida por RNA , Metabolismo , Ribonuclease H , Metabolismo , Tiofenos , Farmacologia , Replicação Viral
4.
Acta Pharmaceutica Sinica ; (12): 1011-1016, 2012.
Artigo em Chinês | WPRIM | ID: wpr-276207

RESUMO

This study is to investigate the mechanism of action of lindenane disesquiterpenoid shizukaol F on HIV-1 replication. Real time quantity PCR, ELISA assay and fluorescence methods were used to test HIV-1 reverse transcription process, RNA-dependent DNA polymerase activity, and RNase H activity, respectively. It showed that shizukaol F inhibited LTR/Gag production of HIV-1 reverse transcription with an IC50 of 9.11 micromol x L(-1). This result is consistent with its inhibitory effect on HIV-1 replication (IC50 of 6.12 micromol x L(-1)). Mechanism studies showed that compound shizukaol F inhibited HIV-1 RT-RNase H with IC50 of 26.4 micromol x L(-1), but had no effect on HIV-1 RT RNA-dependent DNA polymerase activity. In conclusion, shizukaol F is a new structural type HIV-1 RNase H inhibitor. This discovery will provide a clue for new type of reverse transcriptase inhibitors development.


Assuntos
Humanos , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas , Química , Farmacologia , Células HEK293 , Transcriptase Reversa do HIV , Metabolismo , HIV-1 , Fisiologia , Concentração Inibidora 50 , Magnoliopsida , Química , Estrutura Molecular , Plantas Medicinais , Química , Leucemia-Linfoma Linfoblástico de Células Precursoras , Patologia , Inibidores da Transcriptase Reversa , Química , Farmacologia , Ribonuclease H , Metabolismo , Sesquiterpenos , Química , Farmacologia , Replicação Viral
5.
Immune Network ; : 16-22, 2004.
Artigo em Coreano | WPRIM | ID: wpr-160488

RESUMO

BACKGROUND: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. METHODS: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. RESULTS: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of 4.46 X 10(9) cfu was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of 4.5 X 10(-7) M and 1.9 X 10(-7) M, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. CONCLUSION: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.


Assuntos
Humanos , Amilose , Anticorpos , Anticorpos Monoclonais , Bacteriófagos , Técnicas de Visualização da Superfície Celular , Células Clonais , Clonagem de Organismos , Terapia Genética , Voluntários Saudáveis , Vírus da Hepatite B , Hepatite B , Hepatite , Hepatócitos , Fragmentos de Imunoglobulinas , Fígado , Proteínas Ligantes de Maltose , Reação em Cadeia da Polimerase , Ribonuclease H , Ribonucleases , RNA , Anticorpos de Cadeia Única
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