Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs ...Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs have been found to play important roles in normal cell physiological activities,and participate in the development of varieties of tumors and other diseases.Previously,we have only been able to determine the function of lncRNAs through multiple mechanisms,including genetic imprinting,chromatin remodeling,splicing regulation,mRNA decay,and translational regulation.Application of technological advances to research into the function of lncRNAs is extremely important.The major tools for exploring lncRNAs include microarrays,RNA sequencing(RNA-seq),Northern blotting,real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR),fluorescence in situ hybridization(FISH),RNA interference(RNAi),RNA-binding protein immunoprecipitation(RIP),chromatin isolation by RNA purification(ChIRP),crosslinking-immunopurification(CLIP),and bioinformatic prediction.In this review,we highlight the functions of lncRNAs,and advanced methods to research lncRNA-protein interactions.展开更多
Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are...Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are required for protection against ferroptosis.The purpose of the present studywas to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis.Methods:Established human lung fibroblasts MRC-5,WI38,and human LUAD H1650,PC9,H1975,H358,A549,and H1299 cell lines,tumor and matched normal adjacent tissues of LUAD,and plasma from healthy individuals and LUAD patients were used in this study.Immunohistochemistry and immunoblotting were used to analyze protein expression,and quantitative reverse transcription-PCR was used to analyze mRNA expression.Cell viability,cell death,and the lipid reactive oxygen species generationwere measured to evaluate the responses to ferroptosis.Exosomes were observed using transmission electron microscope.The localization of arachidonic acid(AA)was detected using click chemistry labeling followed by confocal microscopy.Interactions between RNAs and proteins were detected using RNA pull-down,RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation methods.Proteomic analysis was used to investigate RNA-regulated proteins,and metabolomic analysis was performed to analyze metabolites.Cellderived xenograft,patient-derived xenograft,cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism.Results:Plasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis.A potential explanation is that exosomal circRNA_101093(cir93)maintained an elevation in intracellular cir93 in LUAD to modulate AA,a poly-unsaturated fatty acid critical for ferroptosisassociated increased peroxidation in the plasma membrane.Mechanistically,cir93 interacted with and increased fatty acid-binding protei展开更多
RNA editing sites in plant mitochondria and plastids are addressed by pentatricopeptide repeat （PPR） proteins with E or E and DYW domains, which recognize a specific nucleotide motif upstream of the edited nucleotid...RNA editing sites in plant mitochondria and plastids are addressed by pentatricopeptide repeat （PPR） proteins with E or E and DYW domains, which recognize a specific nucleotide motif upstream of the edited nucleotide. In addition, some sites require MORF proteins for efficient RNA editing. Here, we assign the novel E domain-containing PPR protein, MEF13, as being required for editing at eight sites in Arabidopsis thaliana. A SNP in ecotype C24 altering the editing level at only one of the eight target sites was located by genomic mapping. An EMS mutant allele of the gene for MEF13 was identified in a SNaPshot screen of a mutated plant population. At all eight target sites of MEF13, editing levels are reduced in both morf3 and morf8 mutants, but at only one site in morfl mutants, suggesting that specific MEF13-MORF interactions are required. Yeast two-hybrid analyses detect solid connections of MEF13 with MORF1 and weak contact with MORF3 proteins. Yeast three-hybrid （Y3H） analysis shows that the presence of MORF8 enhances the connection between MEF13 and MORF3, suggesting that a MORF3-MORF8 heteromer may form stably or transiently to establish interaction with MEF13.展开更多
Objective:To identify potential drug targets for metastasis colorectal cancer(CRC)patients with low mutational burden by examining differences in immune-related gene expression.Methods:For this study,623 samples were ...Objective:To identify potential drug targets for metastasis colorectal cancer(CRC)patients with low mutational burden by examining differences in immune-related gene expression.Methods:For this study,623 samples were collected from The Cancer Genome Atlas(TCGA)database,comprising tumor mutational burden(TMB),RNA sequencing(RNA-Seq),and clinical data.Differential gene expression analysis,Gene Ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of the identified genes were conducted using the R package.Additionally,a comparative analysis of immune infiltrating cell composition in metastatic and non-metastatic groups was performed.Hub genes,exhibiting high levels of interaction,were selected using the Search Tool for the Retrieval of Interacting Genes/Proteins(STRING)database.The Drug Gene Interaction Database(DGIdb)was then utilized to estimate drugs targeting the identified hub genes.Results:The transcriptome data of 326 colorectal cancer patients with low TMB were analyzed,comprising 58 patients with metastasis and 268 patients without metastasis.Among the differential expression in 1,111 genes for patients with metastasis compared to those without metastasis,733 genes were upregulated,and 378 genes were downregulated.KEGG and GO enrichment analysis indicated significant differences in gene expression in CRC metastatic patients with low TMB compared to non-metastasis patients with low TMB.Enriched pathways included humoral immune response,immunoglobulin production,and regulation of AMPA receptor activity.Two genes related to interleukin-12 were identified through secondary enrichment for immune-related genes.Analysis of tumor-infiltrating immune cell data revealed significant differences in memory-activated T cell CD4 and T cell CD8.Conclusions:This analysis of RNA sequencing data and immune-filtrating cell data revealed significant differences between metastatic colorectal cancer patients with low TMB and their non-metastatic counterparts.These distinctions suggest the possibility o展开更多
病程相关蛋白10(pathogenesis related protein 10,PR10)在植物抵抗病毒侵染中发挥重要作用。前期以甘蔗线条花叶病毒(Sugarcane streak mosaic virus,SCSMV)编码的RNA沉默抑制子P1为诱饵,筛选获得一个甘蔗ShPR10蛋白。为探究ShPR10在...病程相关蛋白10(pathogenesis related protein 10,PR10)在植物抵抗病毒侵染中发挥重要作用。前期以甘蔗线条花叶病毒(Sugarcane streak mosaic virus,SCSMV)编码的RNA沉默抑制子P1为诱饵,筛选获得一个甘蔗ShPR10蛋白。为探究ShPR10在甘蔗应答SCSMV侵染过程中的功能,利用同源克隆技术克隆甘蔗ShPR10基因并对其编码蛋白进行生物信息学分析,利用绿色荧光蛋白融合表达法分析ShPR10蛋白的亚细胞定位,采用酵母双杂交和双分子荧光互补技术验证ShPR10与SCSMV P1的互作关系,采用农杆菌共浸润瞬时表达系统和Western blot技术分析ShPR10对P1沉默抑制子活性的影响。结果显示,甘蔗ShPR10基因开放阅读框全长570 bp,编码一个不稳定亲水蛋白,蛋白分子量为21.17 kD,等电点为4.77,含有一个P-loop基序,不含跨膜结构域和信号肽。ShPR10二级结构包含51.85%的无规则卷曲、35.98%的α-螺旋、7.41%的延伸链和4.76%的β-转角。ShPR10蛋白与玉米ZmPR10蛋白的氨基酸序列相似性高达91.53%,两者在进化树上聚为一个分支。ShPR10定位在细胞质和细胞核,与SCSMV P1在酵母细胞和烟草细胞中存在互作关系。ShPR10本身不具有沉默抑制子活性,其表达削弱了P1的沉默抑制子活性,但对P1蛋白的含量无明显影响。综上,ShPR10可能通过结合P1来削弱P1的沉默抑制子活性,从而提高甘蔗对SCSMV的抗性。展开更多
Stress granules(SGs)are cytoplasmic ribonucleoprotein assemblies formed under stress conditions and are related to various biological processes and human diseases.Previous studies have reported the regulatory role of ...Stress granules(SGs)are cytoplasmic ribonucleoprotein assemblies formed under stress conditions and are related to various biological processes and human diseases.Previous studies have reported the regulatory role of some proteins and linear RNAs in SG assembly.However,the relationship between circular RNAs(circRNAs)and SGs has not been discovered.Here,we screened both linear RNAs and circRNAs in SGs using improved total RNA sequencing of purified SG cores in mammalian cells and identified circular transcripts specifically localized in SGs.circRNAs with higher SG-related RNA-binding protein(RBP)binding abilities are more likely to be enriched in SGs.Furthermore,some SG-enriched circRNAs are differentially expressed in hepatocellular carcinoma(HCC)and adjacent tissues.These results suggest the regulatory role of circRNAs in SG formation and provide insights into the biological function of circRNAs and SGs in HCC.展开更多
Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine b...Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.展开更多
基金supported by the National Basic Research Program of China(2010CB912801,2013CB910801)National High Technology Research and Development Program of China(2012AA022501)National Natural Science Foundation of China(31070702,31270836)
文摘Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs have been found to play important roles in normal cell physiological activities,and participate in the development of varieties of tumors and other diseases.Previously,we have only been able to determine the function of lncRNAs through multiple mechanisms,including genetic imprinting,chromatin remodeling,splicing regulation,mRNA decay,and translational regulation.Application of technological advances to research into the function of lncRNAs is extremely important.The major tools for exploring lncRNAs include microarrays,RNA sequencing(RNA-seq),Northern blotting,real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR),fluorescence in situ hybridization(FISH),RNA interference(RNAi),RNA-binding protein immunoprecipitation(RIP),chromatin isolation by RNA purification(ChIRP),crosslinking-immunopurification(CLIP),and bioinformatic prediction.In this review,we highlight the functions of lncRNAs,and advanced methods to research lncRNA-protein interactions.
基金National Natural Science Foundation of China,Grant/Award Numbers:81871907,81822029,81872288,82173015,81902315,81902869,81774291ShanghaiMunicipal Education Commission-Gaofeng Clinical Medicine,Grant/Award Number:20191834+5 种基金Project of Clinical Research Supporting SystemClinical Medicine First-class DisciplineShanghai Municipal Education Commission and Shanghai Education Development Foundation,Grant/Award Number:18CG16Shanghai Sailing Program,Grant/Award Number:19YF1444800Science and technology commission of Shanghai municipality project,Grant/Award Numbers:19140902600,21140902800Shanghai ChestHospital,Grant/Award Numbers:2018YNJCM01,2019YNJCM06,2021YNZYJ01,2021YNZYY01,2021YNZYY02。
文摘Background:Resistance to ferroptosis,a regulated cell death caused by irondependent excessive accumulation of lipid peroxides,has recently been linked to lung adenocarcinoma(LUAD).Intracellular antioxidant systems are required for protection against ferroptosis.The purpose of the present studywas to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis.Methods:Established human lung fibroblasts MRC-5,WI38,and human LUAD H1650,PC9,H1975,H358,A549,and H1299 cell lines,tumor and matched normal adjacent tissues of LUAD,and plasma from healthy individuals and LUAD patients were used in this study.Immunohistochemistry and immunoblotting were used to analyze protein expression,and quantitative reverse transcription-PCR was used to analyze mRNA expression.Cell viability,cell death,and the lipid reactive oxygen species generationwere measured to evaluate the responses to ferroptosis.Exosomes were observed using transmission electron microscope.The localization of arachidonic acid(AA)was detected using click chemistry labeling followed by confocal microscopy.Interactions between RNAs and proteins were detected using RNA pull-down,RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation methods.Proteomic analysis was used to investigate RNA-regulated proteins,and metabolomic analysis was performed to analyze metabolites.Cellderived xenograft,patient-derived xenograft,cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism.Results:Plasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis.A potential explanation is that exosomal circRNA_101093(cir93)maintained an elevation in intracellular cir93 in LUAD to modulate AA,a poly-unsaturated fatty acid critical for ferroptosisassociated increased peroxidation in the plasma membrane.Mechanistically,cir93 interacted with and increased fatty acid-binding protei
文摘RNA editing sites in plant mitochondria and plastids are addressed by pentatricopeptide repeat （PPR） proteins with E or E and DYW domains, which recognize a specific nucleotide motif upstream of the edited nucleotide. In addition, some sites require MORF proteins for efficient RNA editing. Here, we assign the novel E domain-containing PPR protein, MEF13, as being required for editing at eight sites in Arabidopsis thaliana. A SNP in ecotype C24 altering the editing level at only one of the eight target sites was located by genomic mapping. An EMS mutant allele of the gene for MEF13 was identified in a SNaPshot screen of a mutated plant population. At all eight target sites of MEF13, editing levels are reduced in both morf3 and morf8 mutants, but at only one site in morfl mutants, suggesting that specific MEF13-MORF interactions are required. Yeast two-hybrid analyses detect solid connections of MEF13 with MORF1 and weak contact with MORF3 proteins. Yeast three-hybrid （Y3H） analysis shows that the presence of MORF8 enhances the connection between MEF13 and MORF3, suggesting that a MORF3-MORF8 heteromer may form stably or transiently to establish interaction with MEF13.
文摘Objective:To identify potential drug targets for metastasis colorectal cancer(CRC)patients with low mutational burden by examining differences in immune-related gene expression.Methods:For this study,623 samples were collected from The Cancer Genome Atlas(TCGA)database,comprising tumor mutational burden(TMB),RNA sequencing(RNA-Seq),and clinical data.Differential gene expression analysis,Gene Ontology(GO),and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of the identified genes were conducted using the R package.Additionally,a comparative analysis of immune infiltrating cell composition in metastatic and non-metastatic groups was performed.Hub genes,exhibiting high levels of interaction,were selected using the Search Tool for the Retrieval of Interacting Genes/Proteins(STRING)database.The Drug Gene Interaction Database(DGIdb)was then utilized to estimate drugs targeting the identified hub genes.Results:The transcriptome data of 326 colorectal cancer patients with low TMB were analyzed,comprising 58 patients with metastasis and 268 patients without metastasis.Among the differential expression in 1,111 genes for patients with metastasis compared to those without metastasis,733 genes were upregulated,and 378 genes were downregulated.KEGG and GO enrichment analysis indicated significant differences in gene expression in CRC metastatic patients with low TMB compared to non-metastasis patients with low TMB.Enriched pathways included humoral immune response,immunoglobulin production,and regulation of AMPA receptor activity.Two genes related to interleukin-12 were identified through secondary enrichment for immune-related genes.Analysis of tumor-infiltrating immune cell data revealed significant differences in memory-activated T cell CD4 and T cell CD8.Conclusions:This analysis of RNA sequencing data and immune-filtrating cell data revealed significant differences between metastatic colorectal cancer patients with low TMB and their non-metastatic counterparts.These distinctions suggest the possibility o
基金supported by grants from the National Key R&D Program of China(Grant Nos.2021YFA1300500 and 2021YFA1302000)the National Natural Science Foundation of China(Grant Nos.32130020,32025009,and 91940306).
文摘Stress granules(SGs)are cytoplasmic ribonucleoprotein assemblies formed under stress conditions and are related to various biological processes and human diseases.Previous studies have reported the regulatory role of some proteins and linear RNAs in SG assembly.However,the relationship between circular RNAs(circRNAs)and SGs has not been discovered.Here,we screened both linear RNAs and circRNAs in SGs using improved total RNA sequencing of purified SG cores in mammalian cells and identified circular transcripts specifically localized in SGs.circRNAs with higher SG-related RNA-binding protein(RBP)binding abilities are more likely to be enriched in SGs.Furthermore,some SG-enriched circRNAs are differentially expressed in hepatocellular carcinoma(HCC)and adjacent tissues.These results suggest the regulatory role of circRNAs in SG formation and provide insights into the biological function of circRNAs and SGs in HCC.
基金supported by the National Natural Science Foundation of China (21025521, 21035001&20875027)the National Key Basic Re-search Program (2011CB911000)+3 种基金European Commission FP7-HEALTH-2010 Programme-GlycoHIT (260600)National Grand Program on Key Infectious Disease (2009ZX10004-312)Postdoctoral Science Foundation (20100480934) of ChinaChangjiang Scholars and Innovative Research Team in University Program and Natural Science Foundation of Hunan Province (10JJ7002)
文摘Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.
