Identification of the splice sites is a critical and tough issue in eukaryotic genome annotation. Here, a statistical study is introduced for detecting the splicing signals in the human hemoglobin （Hb） pre-mRNAs by ...Identification of the splice sites is a critical and tough issue in eukaryotic genome annotation. Here, a statistical study is introduced for detecting the splicing signals in the human hemoglobin （Hb） pre-mRNAs by using the approaches of regional pairwise alignment, splicing weight matrix scoring, and dynamic extended folding. First, the regional pairwise alignment results show that the coding regions of the human Hb genes are at a high level for both conservation and fluctuation. Second, the weighted matrix scoring results indicate that, although the authentic splicing motifs are always scored the highest in a sequence, the sequence motif alone is inadequate to precisely define the splice sites. Finally, we deduce the RNA frame structures by applying an extended folding approach to analyze the stable folding elements. We find out that the splice sequences tend to take stretching and partially paired conformations, which benefit recognition and competitive binding of the splicing factors. These results indicate that precise splicing is an integrated effect of multiple mechanisms of signal recognition at the level of sequence and structure.展开更多
In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats a...In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.展开更多
Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNAPhe precursors with different nucleotides 37 and 38 were...Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNAPhe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease. The precursors with purine nudeolides at N37 and N38 were found to be the best substrates for the enzyme. When N37 and N38 were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease. If one is pyrimidine nucleotide, the other one is purine nudeotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above. The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.展开更多
Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regu...Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.展开更多
The firmness of table grape berries is a crucial quality parameter. Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive. To enhance our comprehension of the under...The firmness of table grape berries is a crucial quality parameter. Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive. To enhance our comprehension of the underlying molecular factors, we initially identified differentially expressed genes(DEGs) by comparing the transcriptomes of folic acid(FA)-treated and water-treated(CK) berries at different time points. We then analyzed the sequences to detect alternatively spliced(AS) genes associated with postharvest softening. A total of 2,559 DEGs were identified and categorized into four subclusters based on their expression patterns, with subcluster-4 genes exhibiting higher expression in the CK group compared with the FA treatment group. There were 1,045 AS-associated genes specific to FA-treated berries and 1,042 in the CK-treated berries, respectively. Gene Ontology(GO) annotation indicated that the AS-associated genes in CK-treated berries were predominantly enriched in cell wall metabolic processes,particularly cell wall degradation processes. Through a comparison between treatment-associated AS genes and subcluster-4 DEGs, we identified eight genes, including Pectinesterase 2(VvPE2, Vitvi15g00704), which encodes a cell wall-degrading enzyme and was predicted to undergo an A3SS event. The reverse transcription polymerase chain reaction further confirmed the presence of a truncated transcript variant of VvPE2 in the FA-treated berries.Our study provides a comprehensive analysis of AS events in postharvest grape berries using transcriptome sequencing and underscores the pivotal role of VvPE2 during the postharvest storage of grape berries.展开更多
Background Nurr1 is a member of the nuclear receptor superfamily of transcription factors. The objective of the present study was to identify novel splicing variants of the gene in neuronal and non-neuronal tissues an...Background Nurr1 is a member of the nuclear receptor superfamily of transcription factors. The objective of the present study was to identify novel splicing variants of the gene in neuronal and non-neuronal tissues and determine their functions. Methods Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to screen for Nurr1 splice variants in the adult human central nervous system (CNS) and in other tissues such as lymphocytes,and liver,muscle,and kidney cells. Functional assays of the variants were performed by measuring Nurr1 response element (NuRE) transcriptional activity in vitro . Results In this study,the authors identified a novel splicing variant of Nurr1 within exon 5,found in multiple adult human tissues,including lymphocytes,and liver,muscle,and kidney cells,but not in the brain or spinal cord. Sequencing analysis showed the variant has a 75 bp deletion between nucleotides 1402 and 1476. A functional assay of the Nurr1-c splicing variant,performed by measuring NuRE transcriptional activity in vitro,detected a 39% lower level of luciferase (LUC) activity ( P <0.05).Conclusion A novel splicing variant of Nurr1 exists in human non-neuronal tissues and functional assays suggest that the variant may act as an alternate transcription regulator.展开更多
基金Supported by the National Natural Science Foundation of China (30971454, 9030318, and 90208018)
文摘Identification of the splice sites is a critical and tough issue in eukaryotic genome annotation. Here, a statistical study is introduced for detecting the splicing signals in the human hemoglobin （Hb） pre-mRNAs by using the approaches of regional pairwise alignment, splicing weight matrix scoring, and dynamic extended folding. First, the regional pairwise alignment results show that the coding regions of the human Hb genes are at a high level for both conservation and fluctuation. Second, the weighted matrix scoring results indicate that, although the authentic splicing motifs are always scored the highest in a sequence, the sequence motif alone is inadequate to precisely define the splice sites. Finally, we deduce the RNA frame structures by applying an extended folding approach to analyze the stable folding elements. We find out that the splice sequences tend to take stretching and partially paired conformations, which benefit recognition and competitive binding of the splicing factors. These results indicate that precise splicing is an integrated effect of multiple mechanisms of signal recognition at the level of sequence and structure.
基金supported by the National Basic Research Program of China (973 Program) (No.2006CB943601)
文摘In the alternative splicing, intron retention, of histamine H3 receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5＇ splice site and different 3＇ splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5＇ and 3＇ splice sites on intron retention in HeLa cells. Mutating weak 5＇ and 3＇ splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more sigiaificant for the 3＇ splice site of the longer alternative intron than for the 3＇ splice site of the shorter alternative intron and the common 5＇ splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3＇ splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.
基金Project supported by the National Natural Science Foundation of China.
文摘Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNAPhe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease. The precursors with purine nudeolides at N37 and N38 were found to be the best substrates for the enzyme. When N37 and N38 were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease. If one is pyrimidine nucleotide, the other one is purine nudeotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above. The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.
基金supported by grants from the National Key Research and Development Program of China (2016YFD0101804)the National Natural Science Foundation of China (31788103 and 31420103912)as well as the Chinese Academy of Sciences (QYZDY-SSWSMC030 and GJHZ1602)
文摘Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.
基金financially supported by the National Natural Science Foundation of China(32202560 and 32302470)the Program for Innovative Research Team(in Science and Technology)in University of Henan Province+6 种基金China(21IRTSTHN021)the Natural Science Foundation of HenanChina(232300421112)the Program for Science&Technology Innovation Talents in Universities of Henan ProvinceChina(21HASTIT035)the PhD Research Startup Foundation of Henan University of Science and TechnologyChina(13480068 and 13480067)。
文摘The firmness of table grape berries is a crucial quality parameter. Despite extensive research on postharvest fruit softening, its precise molecular mechanisms remain elusive. To enhance our comprehension of the underlying molecular factors, we initially identified differentially expressed genes(DEGs) by comparing the transcriptomes of folic acid(FA)-treated and water-treated(CK) berries at different time points. We then analyzed the sequences to detect alternatively spliced(AS) genes associated with postharvest softening. A total of 2,559 DEGs were identified and categorized into four subclusters based on their expression patterns, with subcluster-4 genes exhibiting higher expression in the CK group compared with the FA treatment group. There were 1,045 AS-associated genes specific to FA-treated berries and 1,042 in the CK-treated berries, respectively. Gene Ontology(GO) annotation indicated that the AS-associated genes in CK-treated berries were predominantly enriched in cell wall metabolic processes,particularly cell wall degradation processes. Through a comparison between treatment-associated AS genes and subcluster-4 DEGs, we identified eight genes, including Pectinesterase 2(VvPE2, Vitvi15g00704), which encodes a cell wall-degrading enzyme and was predicted to undergo an A3SS event. The reverse transcription polymerase chain reaction further confirmed the presence of a truncated transcript variant of VvPE2 in the FA-treated berries.Our study provides a comprehensive analysis of AS events in postharvest grape berries using transcriptome sequencing and underscores the pivotal role of VvPE2 during the postharvest storage of grape berries.
文摘Background Nurr1 is a member of the nuclear receptor superfamily of transcription factors. The objective of the present study was to identify novel splicing variants of the gene in neuronal and non-neuronal tissues and determine their functions. Methods Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to screen for Nurr1 splice variants in the adult human central nervous system (CNS) and in other tissues such as lymphocytes,and liver,muscle,and kidney cells. Functional assays of the variants were performed by measuring Nurr1 response element (NuRE) transcriptional activity in vitro . Results In this study,the authors identified a novel splicing variant of Nurr1 within exon 5,found in multiple adult human tissues,including lymphocytes,and liver,muscle,and kidney cells,but not in the brain or spinal cord. Sequencing analysis showed the variant has a 75 bp deletion between nucleotides 1402 and 1476. A functional assay of the Nurr1-c splicing variant,performed by measuring NuRE transcriptional activity in vitro,detected a 39% lower level of luciferase (LUC) activity ( P <0.05).Conclusion A novel splicing variant of Nurr1 exists in human non-neuronal tissues and functional assays suggest that the variant may act as an alternate transcription regulator.
