Arabidopsis U6 small nuclear RNA （snRNA） promoters are those transcribed by RNA polymerase Ⅲ, but all the core elements for transcriptional initiation are located in the 5＇ promoter region. Previously, three Arabi...Arabidopsis U6 small nuclear RNA （snRNA） promoters are those transcribed by RNA polymerase Ⅲ, but all the core elements for transcriptional initiation are located in the 5＇ promoter region. Previously, three Arabidopsis U6 snRNA genes （U6-1, U6-26, and U6-29） were identified. Herein, we have further identified three new U6 loci （U6-4, U6-5, and U6-6） in the Arabidopsis genome. Alignment of these revealed that the upstream sequence element and TATA elements were contained in six U6 promoters. In addition, a unique, highly conserved element named the ＂CAT＂ element was observed in the promoter region. To understand the expression patterns of these U6 genes in Arabidopsis, we fused these promoters to the DNA segment of β-glucuronidase and then transferred these six constructs into Arabidopsis. Real-time reverse transcription-polymerase chain reaction analysis of these fused transcripts indicated that the newly identified U6 genes are active in Arabidopsis and that the U6-26 promoter seems to have higher transcriptional activity in leaf, stem, flower and silique. These results help to understand the function of these U6 snRNAs in Arabidopsis.展开更多
Regulation of gene expression at the post-transcriptional level is of crucial importance in the development of an organism. Here we present the characterization of a maize gene, U6 biogenesis-like 1 （UBL1）, which pl...Regulation of gene expression at the post-transcriptional level is of crucial importance in the development of an organism. Here we present the characterization of a maize gene, U6 biogenesis-like 1 （UBL1）, which plays an important role in kernel and seedling development by influencing pre-mRNA splicing. The ubll mutant, exhibiting small kernel and weak seedling, was isolated from a Mutator-tagged population. Trans- genic complementation and three independent mutant alleles confirmed that UBL1, which encodes a putative RNA exonuclease belonging to the 2H phosphodiesterase superfamily, is responsible for the phenotype of ubll. We demonstrated that UBL1 possess the RNA exonuclease activity in vitro and found that loss of UBL1 function in ubll causes decreased level and abnormal 3＇ end constitution of snRNA U6, resulting in splicing defect of mRNAs. Through the in vitro and in vivo studies replacing two histidines with alanines in the H-X-T/S-X （X is a hydrophobic residue） motifs we demonstrated that these two motifs are essential for the normal function of UBL1. We further showed that the function of UBL1 may be conserved across a wide phylogenetic distance as the heterologous expression of maize UBL1 could complement the Arabidopsis ubll mutant.展开更多
RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of ...RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 （GFA1）; a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homologs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogenesis and embryogenesis in plant.展开更多
The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated...The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone m RNAs, m RNA fragments, U6 sn RNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area.展开更多
Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease...Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.展开更多
For abundant expression of antisense RNA, triplex forming RNA and Ribozyme in vivo, a novel vector pBSKneo rU6’ was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet...For abundant expression of antisense RNA, triplex forming RNA and Ribozyme in vivo, a novel vector pBSKneo rU6’ was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet replacing the 61 nt sequence in the middle of U6 snRNA coding region with three restriction enzyme sites. Hela nuclear extract in vitro transcription experiments demonstrated that this vector can effectively express U6 mutant RNA. Containing neo r at the same time, stably transfected pBSKneo rU6’ can be selected easily.展开更多
The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembl...The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4-and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. We have extensively sampled the eukaryotic phylogenetic tree to its deepest roots, but did not find genes potentially encoding either U4-or U6-snRNA in the Giardia and Trichomonas data-bases. Our results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding si展开更多
Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric rib...Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulateTGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme againstTGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions ofboth ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activityin vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozymeinhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. Soit indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the researchon the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidatefor TGFβ1-related diseases.展开更多
U7 small nuclear RNA （snRNA） sequences have been described only for a handful of animal species in the past. Here we describe a computational search for functional U7 snRNA genes throughout vertebrates including the...U7 small nuclear RNA （snRNA） sequences have been described only for a handful of animal species in the past. Here we describe a computational search for functional U7 snRNA genes throughout vertebrates including the upstream sequence elements characteristic for snRNAs transcribed by polymerase Ⅱ. Based on the results of this search, we discuss the high variability of U7 snRNAs in both sequence and structure, and report on an attempt to find U7 snRNA sequences in basal deuterostomes and non-drosophilids insect genomes based on a combination of sequence, structure, and promoter features. Due to the extremely short sequence and the high variability in both sequence and structure, no unambiguous candidates were found. These results cast doubt on putative U7 homologs in even more distant organisms that are reported in the most recent release of the Rfam database.展开更多
基金Supported by the State Key Basic Research and Development Plan of China （2001CB109002）, the National Natural Science Foundation of China （30370893）, Shanghai Municipal Committee of Science and Technology （03JC14061）, the Program for New Century Excellent Talents in University （NCET-04-0403）, and the ShuGuang Scholarship （04SG15）.
文摘Arabidopsis U6 small nuclear RNA （snRNA） promoters are those transcribed by RNA polymerase Ⅲ, but all the core elements for transcriptional initiation are located in the 5＇ promoter region. Previously, three Arabidopsis U6 snRNA genes （U6-1, U6-26, and U6-29） were identified. Herein, we have further identified three new U6 loci （U6-4, U6-5, and U6-6） in the Arabidopsis genome. Alignment of these revealed that the upstream sequence element and TATA elements were contained in six U6 promoters. In addition, a unique, highly conserved element named the ＂CAT＂ element was observed in the promoter region. To understand the expression patterns of these U6 genes in Arabidopsis, we fused these promoters to the DNA segment of β-glucuronidase and then transferred these six constructs into Arabidopsis. Real-time reverse transcription-polymerase chain reaction analysis of these fused transcripts indicated that the newly identified U6 genes are active in Arabidopsis and that the U6-26 promoter seems to have higher transcriptional activity in leaf, stem, flower and silique. These results help to understand the function of these U6 snRNAs in Arabidopsis.
文摘Regulation of gene expression at the post-transcriptional level is of crucial importance in the development of an organism. Here we present the characterization of a maize gene, U6 biogenesis-like 1 （UBL1）, which plays an important role in kernel and seedling development by influencing pre-mRNA splicing. The ubll mutant, exhibiting small kernel and weak seedling, was isolated from a Mutator-tagged population. Trans- genic complementation and three independent mutant alleles confirmed that UBL1, which encodes a putative RNA exonuclease belonging to the 2H phosphodiesterase superfamily, is responsible for the phenotype of ubll. We demonstrated that UBL1 possess the RNA exonuclease activity in vitro and found that loss of UBL1 function in ubll causes decreased level and abnormal 3＇ end constitution of snRNA U6, resulting in splicing defect of mRNAs. Through the in vitro and in vivo studies replacing two histidines with alanines in the H-X-T/S-X （X is a hydrophobic residue） motifs we demonstrated that these two motifs are essential for the normal function of UBL1. We further showed that the function of UBL1 may be conserved across a wide phylogenetic distance as the heterologous expression of maize UBL1 could complement the Arabidopsis ubll mutant.
基金Supported by grants from the Ministry of Science and Technology of China (2007CB108702)the National Natural Science Foundation of China (30425030).
文摘RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 （GFA1）; a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homologs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogenesis and embryogenesis in plant.
基金supported by the National Institutes of Health(GM061146)National Science Foundation(IOS-1340001)the National Natural Science Foundation of China(91440105 and 31571332)
文摘The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone m RNAs, m RNA fragments, U6 sn RNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area.
基金grants from the National Natural Science Foundation of China (91435203 and 31991222)the Major Program of Guangdong Basic and Applied Basic Research (2019B030302006)。
文摘Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.
文摘For abundant expression of antisense RNA, triplex forming RNA and Ribozyme in vivo, a novel vector pBSKneo rU6’ was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet replacing the 61 nt sequence in the middle of U6 snRNA coding region with three restriction enzyme sites. Hela nuclear extract in vitro transcription experiments demonstrated that this vector can effectively express U6 mutant RNA. Containing neo r at the same time, stably transfected pBSKneo rU6’ can be selected easily.
文摘The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4-and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. We have extensively sampled the eukaryotic phylogenetic tree to its deepest roots, but did not find genes potentially encoding either U4-or U6-snRNA in the Giardia and Trichomonas data-bases. Our results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding si
文摘Transforming growth factorβ1 (TGFβ1) is known to be intimately involved inmany cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimerichammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulateTGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme againstTGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions ofboth ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activityin vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozymeinhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. Soit indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the researchon the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidatefor TGFβ1-related diseases.
文摘U7 small nuclear RNA （snRNA） sequences have been described only for a handful of animal species in the past. Here we describe a computational search for functional U7 snRNA genes throughout vertebrates including the upstream sequence elements characteristic for snRNAs transcribed by polymerase Ⅱ. Based on the results of this search, we discuss the high variability of U7 snRNAs in both sequence and structure, and report on an attempt to find U7 snRNA sequences in basal deuterostomes and non-drosophilids insect genomes based on a combination of sequence, structure, and promoter features. Due to the extremely short sequence and the high variability in both sequence and structure, no unambiguous candidates were found. These results cast doubt on putative U7 homologs in even more distant organisms that are reported in the most recent release of the Rfam database.
