Theopen reading regions of ZmPHT1s(inorganic phosphate[Pij transporters)inmaize possess target sites of microRNA399(miR399).However,the relationship between miR399 and ZmPHT1s and its functional importance in response...Theopen reading regions of ZmPHT1s(inorganic phosphate[Pij transporters)inmaize possess target sites of microRNA399(miR399).However,the relationship between miR399 and ZmPHT1s and its functional importance in response to Pi deficiency remain to be explored.We show here that ZmPHT1;1,ZmPHT1;3,and ZmPHT1;13 are the targets of ZmmiRNA399.We found that a long non-coding RNA,PILNCR2(Pi-deficiency-induced IncRNA 2),is transcribed from the opposing DNA strand of ZmPHT1;1 and predominantly localized in the cytoplasm.A ribonuclease protection assay and an RNA-RNA binding assay showed that PILNCR2 and ZmPHT1s could form the RNA/RNA duplexes in vivo and in vitro.A co-expression assay in N.benthamiana revealed that the PILNCR2/ZmPHT1 RNA/RNA duplexes interfere with miR399-guided cleavage of ZmPHT1 mRNAs.Overexpression of PILNCR2 increased low-Pi tolerance in maize,whereas its knockout and knockdown decreased low-Pi tolerance in maize.Consistently,ZmPHT1;3 and ZmPHT1;13 mRNA abundance was increased in transgenic plants overexpressing PILNCR2 but reduced in its knock-out mutants,suggesting that PILNCR2 positively regulates the mRNA abundance of ZmPHT1;3 and ZmPHT1;13 in maize.Collectively,these results indicate that PILNCR2 plays an important role in maize Pihomeostasisby interfering with miRNA399-guided cleavageof ZmPHT1mRNAs.展开更多
Long non-coding RNAs(lncRNAs)are emerging as powerful regulators of adipocyte differentiation,fat metabolism and gene expression.However,the functional roles and mechanisms of lncRNAs in these processes remain unclear...Long non-coding RNAs(lncRNAs)are emerging as powerful regulators of adipocyte differentiation,fat metabolism and gene expression.However,the functional roles and mechanisms of lncRNAs in these processes remain unclear.Here,we identified a novel antisense transcript,named APMAP-AS,transcribed from adipocyte membrane-associated protein(APMAP)in the pig genome.APMAP-AS and APMAP were highly expressed in retroperitoneal adipose of obese pigs,compared with that in control pigs.Using a bone mesenchymal stem cells(BMSCs)adipogenic differentiation model,we found that APMAP-AS positively regulated adipogenic differentiation.APMAP-AS had the potential to form an RNA–RNA duplex with APMAP,and increased the stability of APMAP mRNA.Additionally,APMAP-AS promoted lipid metabolism and inhibited the expression of inflammatory factors.These findings of a natural antisense transcript for a regulatory gene associated with lipid synthesis might further our understanding of lncRNAs in driving adaptive adipose tissue remodeling and preserving metabolic health.展开更多
The first evidence that RNA can function as a regulator of gene expression came from experiments with prokaryotes in the 1980 s. It was shown that Escherichia coli micF isan independent gene,has its own promoter,and e...The first evidence that RNA can function as a regulator of gene expression came from experiments with prokaryotes in the 1980 s. It was shown that Escherichia coli micF isan independent gene,has its own promoter,and encodes a small non-coding RNA that base pairs with and inhibits translation of a target messenger RNA in response to environmental stress conditions. The mic F RNA was isolated,sequenced and shown to be a primary transcript. In vitro experiments showed binding to the target ompF mR NA. Secondary structure probing revealed an imperfect micF RNA/ompF RNA duplex interaction and the presence of a non-canonical base pair. Several transcription factors,including OmpR,regulate micF transcription in response to environmental factors. micF has also been found in other bacterial species,however,recently Gerhart Wagner and J?rg Vogel showed pleiotropic effects and found micF inhibits expression of multiple target mR NAs; importantly,one is the global regulatory gene lrp. In addition,micF RNA was found to interact with its targets in different ways; it either inhibits ribosome binding or induces degradation of the message. Thus the concept and initial experimental evidence that RNA can regulate gene expression was born with prokaryotes.展开更多
RNA molecules play crucial roles in various biological processes. Their regulation and function are mediated by interacting with other molecules. Among them RNA-RNA interactions （RRIs） are important in many basic ce...RNA molecules play crucial roles in various biological processes. Their regulation and function are mediated by interacting with other molecules. Among them RNA-RNA interactions （RRIs） are important in many basic cellular activities including transcription, RNA processing, localization, and translation. However, we just start to unveil the complexity of the knowledge and underlying mechanisms of RRIs. Results： In this review, we will summarize approaches for RRI identifications, including both conventional, focused biophysical and biochemical methods and recently developed large scale sequencing-based techniques. We will also discuss discoveries per RRI type revealed by using these technologies, as well as challenges towards a systematic and functional understanding of RRIs. Conclusions： The development of sequencing-based techniques has revolutionized the study of RRIs. Applying these techniques in multiple organisms has identified thousands of RRls, many of which could potentially regulate multiple aspects of gene expression. However, despite the great breakthrough, the RNA-RNA interactome of any species remains far from complete due to intrinsic complex nature of RRI and limitations in current techniques. More efficient experimental methods and computational framework are needed to obtain the full image of RRI networks, and their possible regulatory roles in biology and medicine.展开更多
The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structura...The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.展开更多
NADH-glutamate dehydrogenase (GDH) is active in human tissues, and is chromatographically purified, and studied because it participates in synthesizing glutamate, a neurotransmitter. But chromatography dissociates the...NADH-glutamate dehydrogenase (GDH) is active in human tissues, and is chromatographically purified, and studied because it participates in synthesizing glutamate, a neurotransmitter. But chromatography dissociates the GDH isoenzymes that synthesize nongenetic code-based RNA enzymes degrading superfluous mRNAs thereby aligning the cellular reactions with the environment of the organism. The aim was to electrophoretically purify human hexameric GDH isoenzymes and to characterize their RNA enzyme synthetic activity as in plants. The outcome could be innovative in chemical dependency diagnosis and management. Multi metrix electrophoresis including free solution isoelectric focusing, and through polyacrylamide and agarose gels were deployed to purify the redox cycle isoenzymes of laryngeal GDH, and to assay their RNA enzyme synthetic activities. The laryngeal GDH displayed the 28 binomial isoenzymes typical of higher organisms. Isoelectric focusing purification produced pure GDH. Redox cycle assays of the GDH isoenzymes produced RNA enzymes that degraded human stomach total RNA. In the reaction mechanism, the Schiff-base intermediate complex between α-ketoglutarate and GDH is the target of nucleophiles, resulting to the disruption of synthesis of glutamate, and RNA enzyme. The strongest nucleophiles are the psychoactive alkaloids of tobacco, cocaine, opium poppy, cannabis smoke because they are capable of reacting with GDH Schiff base intermediate to stimulate synthesis of aberrant RNA enzymes that degrade cohorts of mRNAs thereby changing the biochemical pathways and exacerbating drug overdose and chemical dependency. Electrophoretic purification, and characterization of the RNA enzyme synthetic activity set the forecourt for innovative application of GDH redox cycles in the diagnostic management of chemical dependency.展开更多
An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was trans...An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.展开更多
非编码小RNA(non-coding small RNA,sRNA)是原核生物中最重要的一类转录后调节因子,一般通过碱基互补配对调节靶标基因表达。sRNA及其调控的靶标基因构成了一个复杂庞大的调控网络,在微生物的各个生理过程发挥着关键性的调控作用,包括...非编码小RNA(non-coding small RNA,sRNA)是原核生物中最重要的一类转录后调节因子,一般通过碱基互补配对调节靶标基因表达。sRNA及其调控的靶标基因构成了一个复杂庞大的调控网络,在微生物的各个生理过程发挥着关键性的调控作用,包括毒力和致病性。本文就原核sRNA的产生和功能机制以及sRNA互作网络的鉴定展开综述,以期为发现新的sRNA并阐明它们的调控功能提供新的研究思路。展开更多
基金the National Key Research and Development Program of China(2021YFF1000500)the Agricultural Science and Technology Innovation Program of CAAS to W.-X.L.
文摘Theopen reading regions of ZmPHT1s(inorganic phosphate[Pij transporters)inmaize possess target sites of microRNA399(miR399).However,the relationship between miR399 and ZmPHT1s and its functional importance in response to Pi deficiency remain to be explored.We show here that ZmPHT1;1,ZmPHT1;3,and ZmPHT1;13 are the targets of ZmmiRNA399.We found that a long non-coding RNA,PILNCR2(Pi-deficiency-induced IncRNA 2),is transcribed from the opposing DNA strand of ZmPHT1;1 and predominantly localized in the cytoplasm.A ribonuclease protection assay and an RNA-RNA binding assay showed that PILNCR2 and ZmPHT1s could form the RNA/RNA duplexes in vivo and in vitro.A co-expression assay in N.benthamiana revealed that the PILNCR2/ZmPHT1 RNA/RNA duplexes interfere with miR399-guided cleavage of ZmPHT1 mRNAs.Overexpression of PILNCR2 increased low-Pi tolerance in maize,whereas its knockout and knockdown decreased low-Pi tolerance in maize.Consistently,ZmPHT1;3 and ZmPHT1;13 mRNA abundance was increased in transgenic plants overexpressing PILNCR2 but reduced in its knock-out mutants,suggesting that PILNCR2 positively regulates the mRNA abundance of ZmPHT1;3 and ZmPHT1;13 in maize.Collectively,these results indicate that PILNCR2 plays an important role in maize Pihomeostasisby interfering with miRNA399-guided cleavageof ZmPHT1mRNAs.
基金supported by grants from the National Key R&D Program of China(2021YFA0805903,2020YFA0509500 and 2021YFD1300800)the Sichuan Science and Technology Program,China(2021ZDZX0008,2021YFYZ0009,2021YFYZ0030 and 2022JDJQ0054).
文摘Long non-coding RNAs(lncRNAs)are emerging as powerful regulators of adipocyte differentiation,fat metabolism and gene expression.However,the functional roles and mechanisms of lncRNAs in these processes remain unclear.Here,we identified a novel antisense transcript,named APMAP-AS,transcribed from adipocyte membrane-associated protein(APMAP)in the pig genome.APMAP-AS and APMAP were highly expressed in retroperitoneal adipose of obese pigs,compared with that in control pigs.Using a bone mesenchymal stem cells(BMSCs)adipogenic differentiation model,we found that APMAP-AS positively regulated adipogenic differentiation.APMAP-AS had the potential to form an RNA–RNA duplex with APMAP,and increased the stability of APMAP mRNA.Additionally,APMAP-AS promoted lipid metabolism and inhibited the expression of inflammatory factors.These findings of a natural antisense transcript for a regulatory gene associated with lipid synthesis might further our understanding of lncRNAs in driving adaptive adipose tissue remodeling and preserving metabolic health.
基金support from the Department of Molecular Genetics and Microbiology,Stony Brook University
文摘The first evidence that RNA can function as a regulator of gene expression came from experiments with prokaryotes in the 1980 s. It was shown that Escherichia coli micF isan independent gene,has its own promoter,and encodes a small non-coding RNA that base pairs with and inhibits translation of a target messenger RNA in response to environmental stress conditions. The mic F RNA was isolated,sequenced and shown to be a primary transcript. In vitro experiments showed binding to the target ompF mR NA. Secondary structure probing revealed an imperfect micF RNA/ompF RNA duplex interaction and the presence of a non-canonical base pair. Several transcription factors,including OmpR,regulate micF transcription in response to environmental factors. micF has also been found in other bacterial species,however,recently Gerhart Wagner and J?rg Vogel showed pleiotropic effects and found micF inhibits expression of multiple target mR NAs; importantly,one is the global regulatory gene lrp. In addition,micF RNA was found to interact with its targets in different ways; it either inhibits ribosome binding or induces degradation of the message. Thus the concept and initial experimental evidence that RNA can regulate gene expression was born with prokaryotes.
文摘RNA molecules play crucial roles in various biological processes. Their regulation and function are mediated by interacting with other molecules. Among them RNA-RNA interactions （RRIs） are important in many basic cellular activities including transcription, RNA processing, localization, and translation. However, we just start to unveil the complexity of the knowledge and underlying mechanisms of RRIs. Results： In this review, we will summarize approaches for RRI identifications, including both conventional, focused biophysical and biochemical methods and recently developed large scale sequencing-based techniques. We will also discuss discoveries per RRI type revealed by using these technologies, as well as challenges towards a systematic and functional understanding of RRIs. Conclusions： The development of sequencing-based techniques has revolutionized the study of RRIs. Applying these techniques in multiple organisms has identified thousands of RRls, many of which could potentially regulate multiple aspects of gene expression. However, despite the great breakthrough, the RNA-RNA interactome of any species remains far from complete due to intrinsic complex nature of RRI and limitations in current techniques. More efficient experimental methods and computational framework are needed to obtain the full image of RRI networks, and their possible regulatory roles in biology and medicine.
基金Supported by Spanish Ministry of Economy and Competitiveness,No.BFU2012-31213Junta de Andalucía,No.CVI-7430FEDER funds from the EU
文摘The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus(HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5'-untranslatable regions(5'UTRs) and 3'UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5' terminus of the viral genome and regulated by distal functional RNA domains placed at the 3' end. Subsequent RNA replication strongly depends on the 3'UTR folding and is also influenced by the 5' end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNARNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.
文摘NADH-glutamate dehydrogenase (GDH) is active in human tissues, and is chromatographically purified, and studied because it participates in synthesizing glutamate, a neurotransmitter. But chromatography dissociates the GDH isoenzymes that synthesize nongenetic code-based RNA enzymes degrading superfluous mRNAs thereby aligning the cellular reactions with the environment of the organism. The aim was to electrophoretically purify human hexameric GDH isoenzymes and to characterize their RNA enzyme synthetic activity as in plants. The outcome could be innovative in chemical dependency diagnosis and management. Multi metrix electrophoresis including free solution isoelectric focusing, and through polyacrylamide and agarose gels were deployed to purify the redox cycle isoenzymes of laryngeal GDH, and to assay their RNA enzyme synthetic activities. The laryngeal GDH displayed the 28 binomial isoenzymes typical of higher organisms. Isoelectric focusing purification produced pure GDH. Redox cycle assays of the GDH isoenzymes produced RNA enzymes that degraded human stomach total RNA. In the reaction mechanism, the Schiff-base intermediate complex between α-ketoglutarate and GDH is the target of nucleophiles, resulting to the disruption of synthesis of glutamate, and RNA enzyme. The strongest nucleophiles are the psychoactive alkaloids of tobacco, cocaine, opium poppy, cannabis smoke because they are capable of reacting with GDH Schiff base intermediate to stimulate synthesis of aberrant RNA enzymes that degrade cohorts of mRNAs thereby changing the biochemical pathways and exacerbating drug overdose and chemical dependency. Electrophoretic purification, and characterization of the RNA enzyme synthetic activity set the forecourt for innovative application of GDH redox cycles in the diagnostic management of chemical dependency.
文摘An about 1.5kb functional domain sequence of GCRV-RdRp gene was obtained by using RT-PCR amplification.The amplified fragment was cloned into T7 promoted prokaryotic expression system pRSET-C vector and then was transformed into CaCl 2 treated TOP10F’and BL21(DE3)pLysS competent cells respectively.The recombinants were detected with restriction enzyme digestion and further confirmed the interest insert by sequencing pRSET-C/GCRV-RdRp plasmid,which was in frame with the N-terminal tag and in the proper orientation.SDS-PAGE revealed that the highly expressed fusion protein is produced by inducing with l nm IPTG,and its molecular weight is around 55kD,which is the right size corresponding to the predicted value.It indicated the fused protein was produced in the form of inclusion body with its yield remained steadly more than 60% of total bacterial protein. It also showed that the expressed protein was able to bind immunologically to rabbit anti-GCRV-VP2 serum.
文摘非编码小RNA(non-coding small RNA,sRNA)是原核生物中最重要的一类转录后调节因子,一般通过碱基互补配对调节靶标基因表达。sRNA及其调控的靶标基因构成了一个复杂庞大的调控网络,在微生物的各个生理过程发挥着关键性的调控作用,包括毒力和致病性。本文就原核sRNA的产生和功能机制以及sRNA互作网络的鉴定展开综述,以期为发现新的sRNA并阐明它们的调控功能提供新的研究思路。
