We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcri...We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.展开更多
Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature...Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro, 32p labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide （EB） staining. As a result, although there are more unspecific bands in the EB staining assay than 32p labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.展开更多
In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) splicing.Alternative splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a si...In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) splicing.Alternative splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a single pre-mRNA,resulting in functionally distinct proteins.Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression.Mis-regulation of splicing causes a wide range of human diseases.This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing.It also discusses emerging directions in the field of alternative splicing.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
Background： Cellular non-coding RNAs are extensively modified post-transcriptionally, with more than 100 chemically distinct nucleotides identified to date. In the past five years, new sequencing based methods have r...Background： Cellular non-coding RNAs are extensively modified post-transcriptionally, with more than 100 chemically distinct nucleotides identified to date. In the past five years, new sequencing based methods have revealed widespread decoration of eukaryotic messenger RNA with diverse RNA modifications whose functions in mRNA metabolism are only beginning to be known. Results： Since most of the identified mRNA modifying enzymes are present in the nucleus, these modifications have the potential to function in nuclear pre-mRNA processing including alternative splicing. Here we review recent progress towards illuminating the role of pre-mRNA modifications in splicing and highlight key areas for future investigation in this rapidly growing field. Conclusions： Future studies to identify which modifications are added to nascent pre-mRNA and to interrogate the direct effects of individual modifications are likely to reveal new mechanisms by which nuclear pre-mRNA processing is regulated.展开更多
The objective of the present study is to establish a minigene model for studying pre-mRNA alternative splicing. To prepare the minigene DNA constructs, with human or mouse genomic DNA as templates, GluR-B , FGF-2R and...The objective of the present study is to establish a minigene model for studying pre-mRNA alternative splicing. To prepare the minigene DNA constructs, with human or mouse genomic DNA as templates, GluR-B , FGF-2R and Zis 搈inigene?fragments were amplified us-ing PCR and cloned to the eukaryotic expression vectors. The three constructed minigenes and the expression vectors of Tra2b1 and Zis2 were co-transfected in Hela cells. RT-PCR analysis was performed to semi-quantitatively determine the spliced products from the minigenes. The results demonstrated that the constructed minigenes are useful in studying the pre-mRNA al-ternative splicing in cultured cells. With the established Zis minigene, we for the first time found that Zis2 isoform regulates the alternative splicing of Zis minigene.展开更多
Protein arginine methylation plays important roles in diverse biological processes, but its role in regulating shoot regeneration remains elusive. In this study, we characterized the function of the protein arginine m...Protein arginine methylation plays important roles in diverse biological processes, but its role in regulating shoot regeneration remains elusive. In this study, we characterized the function of the protein arginine methyltransferase AtPRMT5 during de novo shoot regeneration in Arabidopsis. AtPRMT5 encodes a type II protein arginine methyltransferase that methylates proteins, including histories and RNA splicing factors. The frequency of shoot regeneration and the number of shoots per callus were decreased in the atprmt5 mutant compared with those in the wild type. Chromatin immunoprecipitation analysis revealed that AtPRMT5 targets KIP-RELATED PROTEINs （KRPs）, which encode the cyclin-dependent kinase inhibitors that repress the cell cycle. During shoot regeneration, the KRP transcript level increased in the atprmt5 mutant, which resulted from reduced histone H4R3 methylation in the KRP promoter. Overexpression of KRP significantly reduced the frequency of shoot regeneration and shoot number per callus. Furthermore, abnormal pre-mRNA splicing in the gene RELATED TO KPC1 （RKP）, which encodes an ubiquitin E3 ligase, was detected in the atprmt5 mutant. RKP functions in regulating KRP protein degradation, and mutation in RKP inhibited shoot regeneration. Thus, AtPRMT5 regulated shoot regeneration through histone modification-mediated KRP transcription and RKP pre-mRNA splicing. Our findings provide new insights into the function of protein arginine methylation in de novo shoot regeneration.展开更多
Pre-mRNA(messenger RNA)splicing participates in the regulation of numerous biological processes in plants.For example,alternative splicing shapes transcriptomic responses to abiotic and biotic stress,and controls deve...Pre-mRNA(messenger RNA)splicing participates in the regulation of numerous biological processes in plants.For example,alternative splicing shapes transcriptomic responses to abiotic and biotic stress,and controls developmental programs.However,no study has revealed a role for splicing in maintaining the root stem cell niche.Here,a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre-m RNA splicing(rdm16-4).The rdm16-4 mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem.The PLETHORA1(PLT1)and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16-4 mutants,resulting in a disordered root stem cell niche and retarded root growth.The root cap of rdm16-4 contained reduced levels of cytokinins,which promote differentiation in the developing root.This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors,such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS(ARR1,ARR2,and ARR11).Furthermore,expression of the full-length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short-root phenotype of rdm16-4.This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.展开更多
Pre-mRNA splicing is a dynamic process. It is catalyzed by the spliceosome which is a large machine formed by an ordered interactions of several small nuclear ribonucleoproteins, U1,
文摘We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.
基金We thank Hao Zhang for technical assistance, Dr. Robin Reed for providing AdML plasmids. The work was supported by grants from National Natural Science Foundation of China （30670441, 30300070）Program for New Century Excellent Talents in University （NCET-04-0245）+1 种基金 Specialized Fund for the Doctoral Program of Higher Education （20040062003）Tianjin Municipal Science and Technology Commission （043802811）.
文摘Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome that catalyzes the removal of non-coding intron sequences to ligate exons into mature mRNA prior to transport and translation. The purpose of our study is to explore whether the in vitro unlabeled pre-mRNA splicing assay could be performed as an alternative method of splicing reaction other than the radiolabeled one. Two different splicing methods in vitro, 32p labeled and unlabeled pre-mRNA as the substrates in the reaction, were investigated. The radiolabeled products were visualized by autoradiography while the unlabeled products were observed by Ethidium Bromide （EB） staining. As a result, although there are more unspecific bands in the EB staining assay than 32p labeled one, the RNA products of in vitro splicing could be observed clearly. This suggests that the unlabeled pre-mRNA splicing assay can be an optional substitution for the isotope-labeled assay.
基金Supported by the Program of "one Hundred Talented people" of the Chinese Academy of Sciences
文摘In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) splicing.Alternative splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a single pre-mRNA,resulting in functionally distinct proteins.Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression.Mis-regulation of splicing causes a wide range of human diseases.This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing.It also discusses emerging directions in the field of alternative splicing.
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.
文摘Background： Cellular non-coding RNAs are extensively modified post-transcriptionally, with more than 100 chemically distinct nucleotides identified to date. In the past five years, new sequencing based methods have revealed widespread decoration of eukaryotic messenger RNA with diverse RNA modifications whose functions in mRNA metabolism are only beginning to be known. Results： Since most of the identified mRNA modifying enzymes are present in the nucleus, these modifications have the potential to function in nuclear pre-mRNA processing including alternative splicing. Here we review recent progress towards illuminating the role of pre-mRNA modifications in splicing and highlight key areas for future investigation in this rapidly growing field. Conclusions： Future studies to identify which modifications are added to nascent pre-mRNA and to interrogate the direct effects of individual modifications are likely to reveal new mechanisms by which nuclear pre-mRNA processing is regulated.
基金This work was supported by the National Natural Science Foundation of China(Grant No.30070242)Scientific Foundation for Youth in Life Sciences,Fudan University.
文摘The objective of the present study is to establish a minigene model for studying pre-mRNA alternative splicing. To prepare the minigene DNA constructs, with human or mouse genomic DNA as templates, GluR-B , FGF-2R and Zis 搈inigene?fragments were amplified us-ing PCR and cloned to the eukaryotic expression vectors. The three constructed minigenes and the expression vectors of Tra2b1 and Zis2 were co-transfected in Hela cells. RT-PCR analysis was performed to semi-quantitatively determine the spliced products from the minigenes. The results demonstrated that the constructed minigenes are useful in studying the pre-mRNA al-ternative splicing in cultured cells. With the established Zis minigene, we for the first time found that Zis2 isoform regulates the alternative splicing of Zis minigene.
文摘Protein arginine methylation plays important roles in diverse biological processes, but its role in regulating shoot regeneration remains elusive. In this study, we characterized the function of the protein arginine methyltransferase AtPRMT5 during de novo shoot regeneration in Arabidopsis. AtPRMT5 encodes a type II protein arginine methyltransferase that methylates proteins, including histories and RNA splicing factors. The frequency of shoot regeneration and the number of shoots per callus were decreased in the atprmt5 mutant compared with those in the wild type. Chromatin immunoprecipitation analysis revealed that AtPRMT5 targets KIP-RELATED PROTEINs （KRPs）, which encode the cyclin-dependent kinase inhibitors that repress the cell cycle. During shoot regeneration, the KRP transcript level increased in the atprmt5 mutant, which resulted from reduced histone H4R3 methylation in the KRP promoter. Overexpression of KRP significantly reduced the frequency of shoot regeneration and shoot number per callus. Furthermore, abnormal pre-mRNA splicing in the gene RELATED TO KPC1 （RKP）, which encodes an ubiquitin E3 ligase, was detected in the atprmt5 mutant. RKP functions in regulating KRP protein degradation, and mutation in RKP inhibited shoot regeneration. Thus, AtPRMT5 regulated shoot regeneration through histone modification-mediated KRP transcription and RKP pre-mRNA splicing. Our findings provide new insights into the function of protein arginine methylation in de novo shoot regeneration.
基金supported by grants from the Ministry of Science and Technology of China(2015CB942901 to ZD)the National Natural Science Foundation of China(Projects 31470371 and 31222005 to ZD)+1 种基金Project funded by China Postdoctoral Science Foundation(2018T110683 to BL)the Special Support for Post-doc Creative Funding in Shandong(201701007 to BL)。
文摘Pre-mRNA(messenger RNA)splicing participates in the regulation of numerous biological processes in plants.For example,alternative splicing shapes transcriptomic responses to abiotic and biotic stress,and controls developmental programs.However,no study has revealed a role for splicing in maintaining the root stem cell niche.Here,a screen for defects in root growth in Arabidopsis thaliana identified an ethyl methane sulfonate mutant defective in pre-m RNA splicing(rdm16-4).The rdm16-4 mutant displays a short-root phenotype resulting from fewer cells in the root apical meristem.The PLETHORA1(PLT1)and PLT2 transcription factor genes are important for root development and were alternatively spliced in rdm16-4 mutants,resulting in a disordered root stem cell niche and retarded root growth.The root cap of rdm16-4 contained reduced levels of cytokinins,which promote differentiation in the developing root.This reduction was associated with the alternative splicing of genes encoding cytokinin signaling factors,such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 and ARABIDOPSIS RESPONSE REGULATORS(ARR1,ARR2,and ARR11).Furthermore,expression of the full-length coding sequence of ARR1 or exogenous cytokinin application partially rescued the short-root phenotype of rdm16-4.This reveals that the RDM16-mediated alternative splicing of cytokinin signaling components contributes to root growth.
文摘Pre-mRNA splicing is a dynamic process. It is catalyzed by the spliceosome which is a large machine formed by an ordered interactions of several small nuclear ribonucleoproteins, U1,
