AIM: We have previously demonstrated that cholangiocytes, the epithelial cells lining intrahepatic bile ducts,encode two functional bile acid transporters via alternative splicing of a single gene to facilitate bile a...AIM: We have previously demonstrated that cholangiocytes, the epithelial cells lining intrahepatic bile ducts,encode two functional bile acid transporters via alternative splicing of a single gene to facilitate bile acid vectorial transport. Cholangiocytes possess ASBT,an apical sodium-dependent bile acid transporter to take up bile acids,and t-ASBT,a basolateral alternatively spliced and truncated form of ASBT to efflux bile acids.Though hepatocyte and ileal bile acid transporters are in part regulated by the flux of bile acids, the effect of alterations in bile acid flux on the expression of t-ASBT in terminal ileocytes remains undear.Thus,we tested the hypothesis that expression of ASBT and t-ASBT in cholangiocytes and ileocytes was regulated by bile acid flux. METHODS: Expression of ASBT and t-ASBT message and protein in cholangiocytes and ileocytes isolated from pair- fed rats given control (C) and 1% taurocholate (TCA) or 5% cholestyramine (CY) enriched diets,were assessed by both quantitative RNase protection assays and quantitative immunoblotting.The data obtained from each of the control groups were pooled to reflect the changes observed following TCA and CY treatments with respect to the control diets. Cholangiocyte taurocholate uptake was determined using a novel microperfusion technique on intrahepatic bile duct units (IBDUs) derived from C,TCA and CY fed rats. RESULTS: In cholangiocytes,both ASBT and t-ASBT message RNA and protein were significantly decreased in response to TCA feeding compared to C diet.In contrast, message and protein of both bile acid transporters significantly increased following CY feeding compared to C diet.In the ileum,TCA feeding significantly up-regulated both ASBT and t-ASBT message and protein compared to C diet,while CY feeding significantly down-regulated message and protein of both bile acid transporters compared to C diet.As anticipated from alterations in cholangiocyte ASBT expression,the uptake of taurocholate in microperfused IBDUs derived from rats on TCA diet decreased 2.7-fold,whereas it increased 1.7-fold in those on CY diet compared to C diet fed groups. CONCLUSION: These data demonstrate that expression of ASBT and t-ASBT in cholangiocytes is regulated by a negative feedback loop while the expression of these transporters in terminal ileum is modified via positive feedback.Thus, while transcriptional regulatory mechanisms in response to alterations in bile acid pool size are operative in both cholangiocytes and ileocytes,each cell type responds differently to bile acid supplementation and depletion.展开更多
The pathways leading to synthesis and post-synthetic modification of DNA employed methionine as donor of atoms: the carbon that came from its –CH3 served for DNA replication and repair either in bacteria or humans;it...The pathways leading to synthesis and post-synthetic modification of DNA employed methionine as donor of atoms: the carbon that came from its –CH3 served for DNA replication and repair either in bacteria or humans;its entire –CH3 served instead for building N6-methyladenine and 5-methylcytosine on bacterial DNA and 5-methylcytosine alone on human DNA. In humans, although a slight extra-S asymmetric methylation appeared de novo yielding on parental DNA 5’-m5CpC-3’/ 3’-GpG-5’, 5’-m5CpT-3’/3’-GpA-5’ and 5’-m5CpA-3’/3’-GpT-5’ monomethylated dinucleotide pairs, a heavy symmetric methylation involved in S semiconservatively newly made DNA to guarantee genetic maintenance of –CH3 in 5’-m5CpG-3’/3’-Gpm5C-5’ dimethylated dinucleotide pairs. In this framework, an inverse correlation was found between bulk genomic DNA methylation occurring in S and bulk polyA-containing pre-mRNA transcription taking place in G1 and G2. Thus, probes of 1 × 106 Daltons (constructed using sheared by sonication newly made methylated DNA filaments) revealed a modular organization in genes: after the hypermethylated promoter, they exhibited an alternation of unmethylated coding and methylated uncoding sequences. This encouraged the search for a language that genes regulated by methylation should have in common. An initial deciphering of restriction minimaps with hypomethylatable exons vs. hypermethylatable promoters and introns was improved when the bisulfite technique allowed a direct sequencing of m5C. In lymphocytes, where the transglutaminase gene is inactive, its promoter exhibited two fully methylated CpG-rich domains at 5’ and one fully unmethylated CpG-rich domain at 3’, including the site +1 and a 5’-UTR. At variance, in HUVEC cells, where the transglutaminase gene is active, in the first CpG-rich domain of promoter few doublets lost their –CH3. Such an inverse correlation suggested new hypotheses especially in connection with repair-modification: UV radiation would cause demethylation in given loci of a promoter by chance, whilst even a partial demethylation in this promoter would be able to resume a previously silent pre-mRNA transcription.展开更多
The intron is an important component of eu-karyotic gene. Extensive studies have been conducted to get a better understanding of its structure and function. This paper presents a brief review of the structure and func...The intron is an important component of eu-karyotic gene. Extensive studies have been conducted to get a better understanding of its structure and function. This paper presents a brief review of the structure and function of introns in higher plant genes. It is shown that higher plant introns possess structural properties shared by all eukaryotic introns, however, they also exhibit a striking degree of diversity. The process of intron splicing in higher plant genes involves interaction between multiple cis -acting elements and trans-acting factors, such as 5’ splicing site, 3’ splicing site and many protein factors. The process of intron splicing is an important level at which gene expression is regulated. Especially alternative splicing of intron can regulate time and space of gene expression. In addition, some introns in higher plant genes also regulate gene expression by affecting the pattern of gene expression, enhancing the level of gene expression and driving the gene expression.展开更多
GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address th...GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.展开更多
Intron Retention(IR)is an alternative splicing mode through which introns are retained in mature RNAs rather than being spliced in most cases.IR has been gaining increasing attention in recent years because of its rec...Intron Retention(IR)is an alternative splicing mode through which introns are retained in mature RNAs rather than being spliced in most cases.IR has been gaining increasing attention in recent years because of its recognized association with gene expression regulation and complex diseases.Continuous efforts have been dedicated to the development of IR detection methods.These methods differ in their metrics to quantify retention propensity,performance to detect IR events,functional enrichment of detected IRs,and computational speed.A systematic experimental comparison would be valuable to the selection and use of existing methods.In this work,we conduct an experimental comparison of existing IR detection methods.Considering the unavailability of a gold standard dataset of intron retention,we compare the IR detection performance on simulation datasets.Then,we compare the IR detection results with real RNA-Seq data.We also describe the use of differential analysis methods to identify disease-associated IRs and compare differential IRs along with their Gene Ontology enrichment,which is illustrated on an Alzheimer’s disease RNA-Seq dataset.We discuss key principles and features of existing approaches and outline their differences.This systematic analysis provides helpful guidance for interrogating transcriptomic data from the point of view of IR.展开更多
文摘AIM: We have previously demonstrated that cholangiocytes, the epithelial cells lining intrahepatic bile ducts,encode two functional bile acid transporters via alternative splicing of a single gene to facilitate bile acid vectorial transport. Cholangiocytes possess ASBT,an apical sodium-dependent bile acid transporter to take up bile acids,and t-ASBT,a basolateral alternatively spliced and truncated form of ASBT to efflux bile acids.Though hepatocyte and ileal bile acid transporters are in part regulated by the flux of bile acids, the effect of alterations in bile acid flux on the expression of t-ASBT in terminal ileocytes remains undear.Thus,we tested the hypothesis that expression of ASBT and t-ASBT in cholangiocytes and ileocytes was regulated by bile acid flux. METHODS: Expression of ASBT and t-ASBT message and protein in cholangiocytes and ileocytes isolated from pair- fed rats given control (C) and 1% taurocholate (TCA) or 5% cholestyramine (CY) enriched diets,were assessed by both quantitative RNase protection assays and quantitative immunoblotting.The data obtained from each of the control groups were pooled to reflect the changes observed following TCA and CY treatments with respect to the control diets. Cholangiocyte taurocholate uptake was determined using a novel microperfusion technique on intrahepatic bile duct units (IBDUs) derived from C,TCA and CY fed rats. RESULTS: In cholangiocytes,both ASBT and t-ASBT message RNA and protein were significantly decreased in response to TCA feeding compared to C diet.In contrast, message and protein of both bile acid transporters significantly increased following CY feeding compared to C diet.In the ileum,TCA feeding significantly up-regulated both ASBT and t-ASBT message and protein compared to C diet,while CY feeding significantly down-regulated message and protein of both bile acid transporters compared to C diet.As anticipated from alterations in cholangiocyte ASBT expression,the uptake of taurocholate in microperfused IBDUs derived from rats on TCA diet decreased 2.7-fold,whereas it increased 1.7-fold in those on CY diet compared to C diet fed groups. CONCLUSION: These data demonstrate that expression of ASBT and t-ASBT in cholangiocytes is regulated by a negative feedback loop while the expression of these transporters in terminal ileum is modified via positive feedback.Thus, while transcriptional regulatory mechanisms in response to alterations in bile acid pool size are operative in both cholangiocytes and ileocytes,each cell type responds differently to bile acid supplementation and depletion.
文摘The pathways leading to synthesis and post-synthetic modification of DNA employed methionine as donor of atoms: the carbon that came from its –CH3 served for DNA replication and repair either in bacteria or humans;its entire –CH3 served instead for building N6-methyladenine and 5-methylcytosine on bacterial DNA and 5-methylcytosine alone on human DNA. In humans, although a slight extra-S asymmetric methylation appeared de novo yielding on parental DNA 5’-m5CpC-3’/ 3’-GpG-5’, 5’-m5CpT-3’/3’-GpA-5’ and 5’-m5CpA-3’/3’-GpT-5’ monomethylated dinucleotide pairs, a heavy symmetric methylation involved in S semiconservatively newly made DNA to guarantee genetic maintenance of –CH3 in 5’-m5CpG-3’/3’-Gpm5C-5’ dimethylated dinucleotide pairs. In this framework, an inverse correlation was found between bulk genomic DNA methylation occurring in S and bulk polyA-containing pre-mRNA transcription taking place in G1 and G2. Thus, probes of 1 × 106 Daltons (constructed using sheared by sonication newly made methylated DNA filaments) revealed a modular organization in genes: after the hypermethylated promoter, they exhibited an alternation of unmethylated coding and methylated uncoding sequences. This encouraged the search for a language that genes regulated by methylation should have in common. An initial deciphering of restriction minimaps with hypomethylatable exons vs. hypermethylatable promoters and introns was improved when the bisulfite technique allowed a direct sequencing of m5C. In lymphocytes, where the transglutaminase gene is inactive, its promoter exhibited two fully methylated CpG-rich domains at 5’ and one fully unmethylated CpG-rich domain at 3’, including the site +1 and a 5’-UTR. At variance, in HUVEC cells, where the transglutaminase gene is active, in the first CpG-rich domain of promoter few doublets lost their –CH3. Such an inverse correlation suggested new hypotheses especially in connection with repair-modification: UV radiation would cause demethylation in given loci of a promoter by chance, whilst even a partial demethylation in this promoter would be able to resume a previously silent pre-mRNA transcription.
文摘The intron is an important component of eu-karyotic gene. Extensive studies have been conducted to get a better understanding of its structure and function. This paper presents a brief review of the structure and function of introns in higher plant genes. It is shown that higher plant introns possess structural properties shared by all eukaryotic introns, however, they also exhibit a striking degree of diversity. The process of intron splicing in higher plant genes involves interaction between multiple cis -acting elements and trans-acting factors, such as 5’ splicing site, 3’ splicing site and many protein factors. The process of intron splicing is an important level at which gene expression is regulated. Especially alternative splicing of intron can regulate time and space of gene expression. In addition, some introns in higher plant genes also regulate gene expression by affecting the pattern of gene expression, enhancing the level of gene expression and driving the gene expression.
基金foundations from Chinese Academy of Sciences and Special Funds for Major State Basic reseaxch of China (G1999053903).
文摘GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5’ Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5’RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5’ direction.
基金supported by the National Natural Science Foundation of China(Nos.61772556,61972185,U1909208,61972423,and 61832019)111 Project(No.B18059)+1 种基金Hunan Provincial Science and Technology Program(No.2018WK4001)Support for these studies was provided by the NIH U01 AG046139
文摘Intron Retention(IR)is an alternative splicing mode through which introns are retained in mature RNAs rather than being spliced in most cases.IR has been gaining increasing attention in recent years because of its recognized association with gene expression regulation and complex diseases.Continuous efforts have been dedicated to the development of IR detection methods.These methods differ in their metrics to quantify retention propensity,performance to detect IR events,functional enrichment of detected IRs,and computational speed.A systematic experimental comparison would be valuable to the selection and use of existing methods.In this work,we conduct an experimental comparison of existing IR detection methods.Considering the unavailability of a gold standard dataset of intron retention,we compare the IR detection performance on simulation datasets.Then,we compare the IR detection results with real RNA-Seq data.We also describe the use of differential analysis methods to identify disease-associated IRs and compare differential IRs along with their Gene Ontology enrichment,which is illustrated on an Alzheimer’s disease RNA-Seq dataset.We discuss key principles and features of existing approaches and outline their differences.This systematic analysis provides helpful guidance for interrogating transcriptomic data from the point of view of IR.
