Lipid droplets （LDs） are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes ...Lipid droplets （LDs） are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegons. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mrhf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of rnthf-1（RNAi） worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol （TAG） hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.展开更多
F-box proteins are components of the SCF （SkpA-Cullin 1-F-box） E3 ligase complexes, acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation. In humans, at least 22 o...F-box proteins are components of the SCF （SkpA-Cullin 1-F-box） E3 ligase complexes, acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation. In humans, at least 22 out of 75 F-box proteins have experimentally documented substrates, whereas in Drosophila 12 F-box proteins have been characterized with known substrates. To systematically investigate the genetic and molecular functions of F-box proteins in Drosophila, we performed a survey of the literature and databases. We identified 45 Drosophila genes that encode proteins containing at least one F-box domain. We collected publically available RNAi lines against these genes and used them in a tissue-specific RNAi-based phenotypic screen. Here, we present our systematic phenotypic dataset from the eye, the wing and the notum. This dataset is the first of its kind and represents a useful resource for future studies of the molecular and genetic functions of F-box genes in Drosophila. Our results show that, as expected, F-box genes in Drosophila have regulatory roles in a diverse array of processes including cell proliferation, cell growth, signal transduction, and cellular and animal survival.展开更多
Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too ...Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum（ER） to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site（ERES） protein Tango1.Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically：PH4 a EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and Rab X3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast,led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco（tbc）, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.展开更多
Mycobacterium tuberculosis is the causative agent of tuberculosis(TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is s...Mycobacterium tuberculosis is the causative agent of tuberculosis(TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference(RNAi).This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single-and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.展开更多
Numerous studies indicate that target gene silencing by RNA interference （RNAi） could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi-...Numerous studies indicate that target gene silencing by RNA interference （RNAi） could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi-mediated crop protection. However, there are many limitations using RNAi-based technology for pest control, with the effectiveness target gene selection and reliable double-strand RNA （dsRNA） delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome-scale high-throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro-injection or by feeding as a dietary component. However, micro-injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi-mediated crop protection has been considered as a potential new-generation technology for pest control, or as a complementary method of existing pest control strategies; however, further devel- opment to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi-based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.展开更多
The Zika virus(ZIKV)and dengue virus(DENV)flaviviruses exhibit similar replicative processes but have distinct clinical outcomes.A systematic understanding of virus–host protein–protein interaction networks can reve...The Zika virus(ZIKV)and dengue virus(DENV)flaviviruses exhibit similar replicative processes but have distinct clinical outcomes.A systematic understanding of virus–host protein–protein interaction networks can reveal cellular pathways critical to viral replication and disease pathogenesis.Here we employed three independent systems biology approaches toward this goal.First,protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and20,240 human proteins revealed multiple conserved cellular pathways and protein complexes,including proteasome complexes.Second,an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process.Third,high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds,including six proteasome inhibitors that suppress both ZIKV and DENV replication.Integrative analyses of these orthogonal datasets pinpoint proteasomes as critical host machinery for ZIKV/DENV replication.Our study provides multi-omics datasets for further studies of flavivirus–host interactions,disease pathogenesis,and new drug targets.展开更多
基金supported by the National Key R&D Program of China(No.2016YFA0500100)the National Natural Science Foundation of China(Nos.U1402225,31571388,31671402,31671233,31701018 and 81471082)+1 种基金the"Personalized Medicinesd Molecular Signature-based Drug Discovery and Development",Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDA12030201)the CAS-Croucher Joint Laboratory Project(No.CAS16SC01)
文摘Lipid droplets （LDs） are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegons. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mrhf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of rnthf-1（RNAi） worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol （TAG） hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.
基金financially supported by the National Basic Research Program of China(973 Program)(No.2009CB918702)the National Natural Science Foundation of China(Nos.31071087 and 31100889)
文摘F-box proteins are components of the SCF （SkpA-Cullin 1-F-box） E3 ligase complexes, acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation. In humans, at least 22 out of 75 F-box proteins have experimentally documented substrates, whereas in Drosophila 12 F-box proteins have been characterized with known substrates. To systematically investigate the genetic and molecular functions of F-box proteins in Drosophila, we performed a survey of the literature and databases. We identified 45 Drosophila genes that encode proteins containing at least one F-box domain. We collected publically available RNAi lines against these genes and used them in a tissue-specific RNAi-based phenotypic screen. Here, we present our systematic phenotypic dataset from the eye, the wing and the notum. This dataset is the first of its kind and represents a useful resource for future studies of the molecular and genetic functions of F-box genes in Drosophila. Our results show that, as expected, F-box genes in Drosophila have regulatory roles in a diverse array of processes including cell proliferation, cell growth, signal transduction, and cellular and animal survival.
基金funded by grants from the Natural Science Foundation of China (Nos.31771600 and 31750410689)Tsinghua Initiative Program (No.20131089281)a 1000 Talents award,all to J.C.P.-P
文摘Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix.Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum（ER） to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site（ERES） protein Tango1.Here, we report an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, we examined distribution of GFP-tagged Collagen IV in live animals and found 88 gene hits for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically：PH4 a EFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in our hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and Rab X3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast,led to uncoupling of ERES and Golgi. We additionally present a characterization of a screening hit we named trabuco（tbc）, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, we discuss the success of our screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.
基金supported by the National Key R&D Program of China(Grant No.2017YFD0500303)the National Natural Science Foundation of China(Grant Nos.C180501 and 31602061)+1 种基金the Huazhong Agricultural University Scientific&Technological Self-innovation Foundation,China(Grant Nos.2662017PY105 and 2662017PY105)the Doctoral Fund of Ministry of Education of China(Grant No.131012).
文摘Mycobacterium tuberculosis is the causative agent of tuberculosis(TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type Ⅲ-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference(RNAi).This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single-and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.
文摘Numerous studies indicate that target gene silencing by RNA interference （RNAi） could lead to insect death. This phenomenon has been considered as a potential strategy for insect pest control, and it is termed RNAi-mediated crop protection. However, there are many limitations using RNAi-based technology for pest control, with the effectiveness target gene selection and reliable double-strand RNA （dsRNA） delivery being two of the major challenges. With respect to target gene selection, at present, the use of homologous genes and genome-scale high-throughput screening are the main strategies adopted by researchers. Once the target gene is identified, dsRNA can be delivered by micro-injection or by feeding as a dietary component. However, micro-injection, which is the most common method, can only be used in laboratory experiments. Expression of dsRNAs directed against insect genes in transgenic plants and spraying dsRNA reagents have been shown to induce RNAi effects on target insects. Hence, RNAi-mediated crop protection has been considered as a potential new-generation technology for pest control, or as a complementary method of existing pest control strategies; however, further devel- opment to improve the efficacy of protection and range of species affected is necessary. In this review, we have summarized current research on RNAi-based technology for pest insect management. Current progress has proven that RNAi technology has the potential to be a tool for designing a new generation of insect control measures. To accelerate its practical application in crop protection, further study on dsRNA uptake mechanisms based on the knowledge of insect physiology and biochemistry is needed.
基金supported by the National Institutes of Health(NIH),USA(Grant Nos.U19AI131130,R01GM111514,R21AI131706,R35NS097370,and R37NS047344)the Intramural Research Program of the NCATS/NIH,USA
文摘The Zika virus(ZIKV)and dengue virus(DENV)flaviviruses exhibit similar replicative processes but have distinct clinical outcomes.A systematic understanding of virus–host protein–protein interaction networks can reveal cellular pathways critical to viral replication and disease pathogenesis.Here we employed three independent systems biology approaches toward this goal.First,protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and20,240 human proteins revealed multiple conserved cellular pathways and protein complexes,including proteasome complexes.Second,an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process.Third,high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds,including six proteasome inhibitors that suppress both ZIKV and DENV replication.Integrative analyses of these orthogonal datasets pinpoint proteasomes as critical host machinery for ZIKV/DENV replication.Our study provides multi-omics datasets for further studies of flavivirus–host interactions,disease pathogenesis,and new drug targets.
