5-Methylcytosine(5mC)plays pivotal roles in numerous biological processes.To gain a deeper understanding of the biological functions of 5mC,it is essential to develop methods for its quantitative analysis.Herein,we en...5-Methylcytosine(5mC)plays pivotal roles in numerous biological processes.To gain a deeper understanding of the biological functions of 5mC,it is essential to develop methods for its quantitative analysis.Herein,we engineered the repressor of silencing 1(ROS1)protein to enhance its glycosylase/lyase activity towards 5mC,resulting in an engineered ROS1(eROS1)protein that can effectively excise 5mC from DNA.Using eROS1,we developed a method termed engineered ROS1-mediated quantitative(eRMQ)analysis,for the locus-specific quantification of 5mC in genomic DNA.This method capitalizes on the ability of eROS1 to selectively cleave 5mC,which creates a one-nucleotide gap.The presence of this gap hinders the extension of DNA polymerase,leading to a reduction in extension products that can be evaluated using real-time quantitative PCR(qPCR).The limit of detection for the eRMQ method was as low as 1 fM.Using the eRMQ method,we achieved the quantitative analysis of 5mC at individual sites within genomic DNA and demonstrated a significant reduction in 5mC levels in lung cancer tissues compared to adjacent normal tissues.Taken together,this study introduces eRMQ method for the quantitative analysis of 5mC in DNA,offering a valuable tool for exploring epigenetic regulation in human diseases.展开更多
In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic spe...In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.展开更多
RNA can interact with RNA-binding proteins(RBPs),mRNA,or other non-coding RNAs(ncRNAs)to form complex regulatory networks.High-throughput CLIP-seq,degradome-seq,and RNA-RNA interactome sequencing methods represent pow...RNA can interact with RNA-binding proteins(RBPs),mRNA,or other non-coding RNAs(ncRNAs)to form complex regulatory networks.High-throughput CLIP-seq,degradome-seq,and RNA-RNA interactome sequencing methods represent powerful approaches to identify biologically relevant ncRNA-target and protein-ncRNA interactions.However,assigning ncRNAs to their regulatory target genes or interacting RNA-binding proteins(RBPs)remains technically challenging.Chemical modifications to mRNA also play important roles in regulating gene expression.Investigation of the functional roles of these modifications relies highly on the detection methods used.RNA structure is also critical at nearly every step of the RNA life cycle.In this review,we summarize recent advances and limitations in CLIP technologies and discuss the computational challenges of and bioinformatics tools used for decoding the functions and regulatory networks of ncRNAs.We also summarize methods used to detect RNA modifications and to probe RNA structure.展开更多
基金supported by the National Natural Science Foundation of China(22277093,22207090)the Fundamental Research Funds for the Central Universities(2042024kf0022)the Key Research and Development Project of Hubei Province(2023BCB094).
文摘5-Methylcytosine(5mC)plays pivotal roles in numerous biological processes.To gain a deeper understanding of the biological functions of 5mC,it is essential to develop methods for its quantitative analysis.Herein,we engineered the repressor of silencing 1(ROS1)protein to enhance its glycosylase/lyase activity towards 5mC,resulting in an engineered ROS1(eROS1)protein that can effectively excise 5mC from DNA.Using eROS1,we developed a method termed engineered ROS1-mediated quantitative(eRMQ)analysis,for the locus-specific quantification of 5mC in genomic DNA.This method capitalizes on the ability of eROS1 to selectively cleave 5mC,which creates a one-nucleotide gap.The presence of this gap hinders the extension of DNA polymerase,leading to a reduction in extension products that can be evaluated using real-time quantitative PCR(qPCR).The limit of detection for the eRMQ method was as low as 1 fM.Using the eRMQ method,we achieved the quantitative analysis of 5mC at individual sites within genomic DNA and demonstrated a significant reduction in 5mC levels in lung cancer tissues compared to adjacent normal tissues.Taken together,this study introduces eRMQ method for the quantitative analysis of 5mC in DNA,offering a valuable tool for exploring epigenetic regulation in human diseases.
基金supported by grants from the Significant Special Found of "13115" S&T Innovation Project of Shaanxi Province,China(2007 ZDKG-01)"13115" Technology Innovation Engineering and Engineering Technology Research Center of Shaanxi Province,China(2008 ZDGC-02)the Special Capital for the Construction of Modern Agriculture Technical System of Shaanxi Province,China (NYCYTX-001)
文摘In the present study, one unique low-molecular-weight glutenin subunit （LMW-GS） gene. LMWXY22-2 （GenBank no. FJ028810）, was isolated from wheat cultivar Xiaoyan 22 （Triticum aestivum L.） by a pair of genomic specific PCR primers for 1B chromosome. Sequence analysis revealed that LMWXY22-2 was composed of 1 364 bp nucleotides, including a 317 bp promotion region and a 1 047 bp coding region which could be translated into a mature protein of 349 amino acids. In spite of a few minor mutations, the sequence of 5＇ untranslated region （UTR）, the coding region, the deduced N- and Cterminus comparisons indicated that LMWXY22-2 belonged to the reported subunits of LMW-m type and type lII group 5, respectively. Inner gene markers for 1D chromosome together with the phylogenetic analysis revealed that this gene was classified into Glu-D3, which was not in agreement with the I B locus-specific primers for LMW genes completely.
文摘RNA can interact with RNA-binding proteins(RBPs),mRNA,or other non-coding RNAs(ncRNAs)to form complex regulatory networks.High-throughput CLIP-seq,degradome-seq,and RNA-RNA interactome sequencing methods represent powerful approaches to identify biologically relevant ncRNA-target and protein-ncRNA interactions.However,assigning ncRNAs to their regulatory target genes or interacting RNA-binding proteins(RBPs)remains technically challenging.Chemical modifications to mRNA also play important roles in regulating gene expression.Investigation of the functional roles of these modifications relies highly on the detection methods used.RNA structure is also critical at nearly every step of the RNA life cycle.In this review,we summarize recent advances and limitations in CLIP technologies and discuss the computational challenges of and bioinformatics tools used for decoding the functions and regulatory networks of ncRNAs.We also summarize methods used to detect RNA modifications and to probe RNA structure.
