OBJECTIVETo synthesize highly pure HBV post-transcriptional regulatory element (HPRE) via transcription in vitro by T7 RNA polymerase.

METHODSHPRE gene was amplified by PCR from a template containing HBV complete genomic DNA and cloned into plasmid pGEM-11zf. The cloned DNA sequence was transcribed by T7 RNA polymerase.

RESULTSThe construction of HPRE gene recombinant plasmid and production of HPRE via transcription in vitro was successful.

CONCLUSIONIn vitro transcription by T7 RNA polymerase can be used to synthesize highly pure HPRE.

DNA-Directed DNA Polymerase ; DNA-Directed RNA Polymerases ; Hepatitis B virus ; genetics ; RNA Processing, Post-Transcriptional ; RNA Splicing ; RNA-Binding Proteins ; physiology ; Transcription, Genetic ; Viral Proteins

The purpose of this review is to confirm the reason resulted the gene silence and explore the countermeasure avoiding the gene silence in transgene plant. The method is to divide the gene silencing into transcriptional gene silencing(TGS) and posttranscriptional gene silencing(PTGS). Several models resulted PTGS were analyzed by RNA threshold model, ectopic pairing and aberrant RNA model and ds-RNA model. The results showed that it was important to decide the phenomena of restraining transgene silencing and the mechanism of PTGS. The strategies of identification of gene function and prevention of virus were presented by RNAi and gene silencing respectively, etc.
Animals ; Gene Silencing ; Models, Genetic ; RNA ; RNA Processing, Post-Transcriptional

OBJECTIVETo analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.

METHODSTwo specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.

RESULTSAfter reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.

CONCLUSIONSCleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.

5' Untranslated Regions ; metabolism ; Base Sequence ; DNA, Catalytic ; genetics ; metabolism ; Hepacivirus ; enzymology ; genetics ; Humans ; Molecular Sequence Data ; RNA Processing, Post-Transcriptional ; RNA, Catalytic ; metabolism ; RNA, Viral ; metabolism ; mRNA Cleavage and Polyadenylation Factors ; genetics ; metabolism

N-methyladenosine (mA) is the most common post-transcriptional RNA modification throughout the transcriptome, affecting fundamental aspects of RNA metabolism. mA modification could be installed by mA "writers" composed of core catalytic components (METTL3/METTL14/WTAP) and newly defined regulators and removed by mA "erasers" (FTO and ALKBH5). The function of mA is executed by mA "readers" that bind to mA directly (YTH domain-containing proteins, eIF3 and IGF2BPs) or indirectly (HNRNPA2B1). In the past few years, advances in mA modulators ("writers," "erasers," and "readers") have remarkably renewed our understanding of the function and regulation of mA in different cells under normal or disease conditions. However, the mechanism and the regulatory network of mA are still largely unknown. Moreover, investigations of the mA physiological roles in human diseases are limited. In this review, we summarize the recent advances in mA research and highlight the functional relevance and importance of mA modification in in vitro cell lines, in physiological contexts, and in cancers.
Adenosine ; analogs & derivatives ; metabolism ; Cell Differentiation ; physiology ; Humans ; Neoplasms ; metabolism ; RNA ; metabolism ; RNA Processing, Post-Transcriptional

The let-7 miRNA was one of the first miRNAs discovered in the nematode, Caenorhabditis elegans, and its biological functions show a high level of evolutionary conservation from the nematode to the human. Unlike in C. elegans, higher animals have multiple isoforms of let-7 miRNAs; these isoforms share a consensus sequence called the 'seed sequence' and these isoforms are categorized into let-7 miRNA family. The expression of let-7 family is required for developmental timing and tumor suppressor function, but must be suppressed for the self-renewal of stem cells. Therefore, let-7 miRNA biogenesis must be carefully controlled. To generate a let-7 miRNA, a primary transcript is produced by RNA polymerase II and then subsequently processed by Drosha/DGCR8, TUTase, and Dicer. Because dysregulation of let-7 processing is deleterious, biogenesis of let-7 is tightly regulated by cellular factors, such as the RNA binding proteins, LIN28A/B and DIS3L2. In this review, we discuss the biological functions and biogenesis of let-7 miRNAs, focusing on the molecular mechanisms of regulation of let-7 biogenesis in vertebrates, such as the mouse and the human.
Animals ; Base Sequence ; Gene Expression Regulation ; Humans ; MicroRNAs ; biosynthesis ; chemistry ; genetics ; metabolism ; RNA Processing, Post-Transcriptional ; RNA Stability ; Transcription, Genetic

TAR DNA binding protein-43(TDP-43) and fused in sarcoma/translocated in liposarcoma protein (FUS/TLS) have been found to be associated with two neurodegenerative diseases - amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in TDP-43 and FUS/TLS lead to abnormal protein expressions, which result in altered RNA processing. The pathological changes of TDP-43 and FUS/TLS-associated ALS and FTD are similar. Although the interactions between ALS and FTD remain unknown, it is speculated that TDP-43 and FUS/TLS-associated neurodegenerative diseases may share similar pathogenesis.
Amyotrophic Lateral Sclerosis ; DNA-Binding Proteins ; genetics ; metabolism ; Frontotemporal Dementia ; Humans ; Mutation ; RNA Processing, Post-Transcriptional ; RNA-Binding Protein FUS ; genetics ; metabolism

Messenger RNA (mRNA) can be modified by more than 100 chemical modifications. Among these modifications, N6-methyladenosine (m⁶A) is one of the most prevalent modifications. During the processes of cells differentiation, embryo development or stress, m⁶A can be modified on key mRNAs and regulate the progress of cells through modulating mRNA metabolism and translation. Other mRNA modifications, including N1-methyladenosine (m¹A), 5-methylcytosine (m⁵C) and pseudouridine, together with m⁶A form the epitranscriptome of mRNA that accurately modulate the mRNA translation. Here we review the types and characteristic of mRNA epigenetic modifications, especially the recent progresses of the function of m⁶A, we also expect the main research direction of m⁶A epigenetic modification in the future.
Adenosine ; analogs & derivatives ; genetics ; metabolism ; Cell Differentiation ; genetics ; Embryonic Development ; genetics ; Epigenesis, Genetic ; Gene Expression Regulation ; RNA Processing, Post-Transcriptional ; RNA, Messenger ; metabolism

The acquisition of somatic mutations is the most common event in cancer. Neoantigens expressed from genes with mutations acquired during carcinogenesis can be tumor-specific. Since the immune system recognizes tumor-specific peptides, they are potential targets for personalized neoantigen-based immunotherapy. However, the discovery of druggable neoantigens remains challenging, suggesting that a deeper understanding of the mechanism of neoantigen generation and better strategies to identify them will be required to realize the promise of neoantigen-based immunotherapy. Alternative splicing and RNA editing events are emerging mechanisms leading to neoantigen production. In this review, we outline recent work involving the large-scale screening of neoantigens produced by alternative splicing and RNA editing. We also describe strategies to predict and validate neoantigens from RNA sequencing data.
Alternative Splicing ; Carcinogenesis ; Humans ; Immune System ; Immunotherapy ; Mass Screening ; Peptides ; RNA Editing ; RNA ; Sequence Analysis, RNA

The relationship between 3 polymorphisms sites [interleulin-4 (IL-4), IL-4 receptor (IL-4R) alpha chain and activation-induced cytidine deaminase (AICDA)] and adult allergic asthma in China was studied. By using case-control method, DNA and clinical data were obtained from allergic asthmatic patients and compared with those in the control subjects. The subjects were genotyped for the IL-4 C-589T promoter polymorphism, the IL-4R alpha chain Q576R and the AICDA C8408T by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The results showed that the IL-4 C-589T was not associated with adult allergic asthma in China. However, the IL-4R alpha chain 576R/R and AICDA 8408T/T frequency was significantly increased in allergic asthma group as compared with that in the control group [odd ratio (OR) = 3.797 and 9.127, respectively; P < 0.01)] and was correlated with the increased plasma total IgE. These data suggested that the IL-4R alpha chain 576R/R and AICDA 8408T/T genotypes confer genetic susceptibility to adult allergic asthma in China.
Adult ; Alleles ; Asthma ; etiology ; genetics ; Cytidine Deaminase ; genetics ; Humans ; Immunoglobulin E ; blood ; Interleukin-4 ; genetics ; Phenotype ; Polymorphism, Restriction Fragment Length ; RNA Processing, Post-Transcriptional ; Receptors, Interleukin-4 ; genetics

The relationship between 3 polymorphisms sites [interleulin-4 (IL-4), IL-4 receptor (IL-4R) alpha chain and activation-induced cytidine deaminase (AICDA)] and adult allergic asthma in China was studied. By using case-control method, DNA and clinical data were obtained from allergic asthmatic patients and compared with those in the control subjects. The subjects were genotyped for the IL-4 C-589T promoter polymorphism, the IL-4R alpha chain Q576R and the AICDA C8408T by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The results showed that the IL-4 C-589T was not associated with adult allergic asthma in China. However, the IL-4R alpha chain 576R/R and AICDA 8408T/T frequency was significantly increased in allergic asthma group as compared with that in the control group [odd ratio (OR) = 3.797 and 9.127, respectively; P < 0.01)] and was correlated with the increased plasma total IgE. These data suggested that the IL-4R alpha chain 576R/R and AICDA 8408T/T genotypes confer genetic susceptibility to adult allergic asthma in China.
Alleles ; Asthma/etiology ; Asthma/*genetics ; Cytidine Deaminase/*genetics ; Immunoglobulin E/blood ; Interleukin-4/*genetics ; Phenotype ; *Polymorphism, Restriction Fragment Length ; RNA Processing, Post-Transcriptional ; Receptors, Interleukin-4/*genetics