Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively e...Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.展开更多
The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study i...The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.展开更多
CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and ...CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin(mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1(mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6 K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6 K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing m TOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells.Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.展开更多
The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genet...The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs（sg RNAs）.Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing（HTS）.We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.展开更多
Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure ter...Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase(P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.展开更多
The International Conference on Arginine and Pyrimidines （ICAP） had its origin as an arginine workshop organised by a group of microbiologists led by Werner Maas in 1972 （Rubio, 2003）. The shared requirement of ca...The International Conference on Arginine and Pyrimidines （ICAP） had its origin as an arginine workshop organised by a group of microbiologists led by Werner Maas in 1972 （Rubio, 2003）. The shared requirement of carbamoyl phosphate in both arginine and pyrimidine metabolism led to a natural association of researchers working in both fields.展开更多
Genome-wide analyses of metazoan messenger RNA （mRNA） species are unveiling the extensive transcriptional diversity generated by alternative splicing （AS）. Research is also beginning to identify the splicing facto...Genome-wide analyses of metazoan messenger RNA （mRNA） species are unveiling the extensive transcriptional diversity generated by alternative splicing （AS）. Research is also beginning to identify the splicing factors and AS events required to maintain the balance between stem cell renewal （i.e stemness properties） and differentiation. One set of proteins at the center of spliceosome biogenesis are the survival motor neuron （SMN） complex constituents, which have a critical role in the assembly of spliceosomal small nuclear ribonucleoproteins （snRNPs） in all cells. In this review we discuss what is currently known about how AS controls pluripotency and cell fate and consider how an increased requirement for splicing factors, including SMN, helps to maintain an enrichment of stem cell-specific AS events. Furthermore, we highlight studies showing that mutations in specific splicing factors can lead to the aberrant development, and cause targeted degeneration of the nervous system. Using SMN as an example, we discuss the perspective of how stem cell-specific changes in splicing factors can lead to developmental defects and the selective degeneration of particular tissues. Finally we consider the expanding role of SMN, and other splicing factors, in the regulation of gene expression in stem cell biology, thereby providing insight into a number of debilitating diseases.展开更多
基金supported by the UK Medical Research Council and the European Research Council (DARCGENs, No. 249869)
文摘Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.
基金supported by the UK Medical Research Council(Grant No.MC_UU_12021/3 and MC_U137788471)University of OxfordShanghaiTech University
文摘The metabolic enzyme CTP synthase(CTPS) is able to compartmentalize into filaments,termed cytoophidia,in a variety of organisms including bacteria,budding yeast,fission yeast,fruit flies and mammals.A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift.Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe.To our surprise,we find that both the length and the occurrence of cytoophidia in S.pombe decrease upon cold shock or heat shock.The temperature-dependent changes of cytoophidia are fast and reversible.Taking advantage of yeast genetics,we demonstrate that heat-shock proteins are required for cytoophidium assembly in S.pombe.Temperature sensitivity of cytoophidia makes S.pombe an attractive model system for future investigations of this novel membraneless organelle.
基金supported by Shanghai Tech University, the National Natural Science Foundation of China (81500266)the UK Medical Research Council (MC_UU_12021/3 and MC_U137788471)
文摘CTP synthase(CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin(mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1(mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6 K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6 K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing m TOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells.Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.
基金the European Research Council(DARCGENs,project number 249869)the Medical Research Council for financial support
文摘The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs（sg RNAs）.Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing（HTS）.We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.
基金supported by ShanghaiTech University,the UK Medical Research Council(Grant No.MC_UU_12021/3 and MC_U137788471)National Natural Science Foundation of China(Grant No.31771490)。
文摘Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism.In 2010,three groups independently reported that CTP synthase(CTPS)can assemble into a filamentous structure termed the cytoophidium.In searching for CTPS-interacting proteins,here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein,△~1-pyrroline-5-carboxylate synthase(P5CS).Using the Drosophila follicle cell as the in vivo model,we confirm that P5CS forms cytoophidia,which are associated with CTPS cytoophidia.Overexpression of P5CS increases the length of CTPS cytoophidia.Conversely,filamentation of CTPS affects the morphology of P5CS cytoophid ia.Finally,in vitro analyses confirm the filament-fo rming property of P5CS.Our work links CTPS with P5CS,two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis,respectively.
基金sponsored by MRC Functional Genomics Unit at the University of Oxford, the Journal of Genetics and Genomics, and Dutscher Scientific
文摘The International Conference on Arginine and Pyrimidines （ICAP） had its origin as an arginine workshop organised by a group of microbiologists led by Werner Maas in 1972 （Rubio, 2003）. The shared requirement of carbamoyl phosphate in both arginine and pyrimidine metabolism led to a natural association of researchers working in both fields.
文摘Genome-wide analyses of metazoan messenger RNA （mRNA） species are unveiling the extensive transcriptional diversity generated by alternative splicing （AS）. Research is also beginning to identify the splicing factors and AS events required to maintain the balance between stem cell renewal （i.e stemness properties） and differentiation. One set of proteins at the center of spliceosome biogenesis are the survival motor neuron （SMN） complex constituents, which have a critical role in the assembly of spliceosomal small nuclear ribonucleoproteins （snRNPs） in all cells. In this review we discuss what is currently known about how AS controls pluripotency and cell fate and consider how an increased requirement for splicing factors, including SMN, helps to maintain an enrichment of stem cell-specific AS events. Furthermore, we highlight studies showing that mutations in specific splicing factors can lead to the aberrant development, and cause targeted degeneration of the nervous system. Using SMN as an example, we discuss the perspective of how stem cell-specific changes in splicing factors can lead to developmental defects and the selective degeneration of particular tissues. Finally we consider the expanding role of SMN, and other splicing factors, in the regulation of gene expression in stem cell biology, thereby providing insight into a number of debilitating diseases.
