本研究通过编辑识别RNA结合蛋白超靶标(hyper targets of RNA-binding proteins identified by editing,HyperTRIBE)技术识别了HEK293细胞中上游移码蛋白1(up-frameshift protein 1,UPF1)的靶基因,并结合UPF1敲低数据研究其关键靶基因...本研究通过编辑识别RNA结合蛋白超靶标(hyper targets of RNA-binding proteins identified by editing,HyperTRIBE)技术识别了HEK293细胞中上游移码蛋白1(up-frameshift protein 1,UPF1)的靶基因,并结合UPF1敲低数据研究其关键靶基因的生物学功能。实验设计采用三组平行质粒转染体系:实验组转染UPF1-ADAR2cd融合表达质粒、对照组转染mcherry-ADAR2cd质粒、背景组转染GFP空载体质粒。转染48 h后收集细胞进行转录组测序,通过HyperTRIBE识别HEK293细胞中UPF1的靶基因,并结合starBase数据库和UPF1敲低数据筛选UPF1关键靶基因。应用基因本体学、京都基因和基因组百科全书分析关键靶基因生物学功能,并使用相互作用基因检索工具绘制靶基因之间的蛋白质相互作用网络。最终,HyperTRIBE成功识别出1606个HEK293细胞中的UPF1靶基因,结合starBase数据库和UPF1敲低数据得到71个关键靶基因。这些靶基因在mRNA监测、细胞内囊泡转运、信号通路调控以及细胞器功能维持等多个生物学过程中发挥重要作用。研究结果证明了HyperTRIBE在识别RNA结合蛋白靶基因方面的有效性和准确性,为UPF1在其他细胞类型中的研究以及其他RNA结合蛋白的功能研究奠定了基础。展开更多
Up-frameshift 1(UPF1),as the most critical factor in nonsense-mediated messenger RNA(mRNA)decay(NMD),regulates tumor-associated molecular pathways in many cancers.However,the role of UPF1 in lung adenocarcinoma(LUAD)a...Up-frameshift 1(UPF1),as the most critical factor in nonsense-mediated messenger RNA(mRNA)decay(NMD),regulates tumor-associated molecular pathways in many cancers.However,the role of UPF1 in lung adenocarcinoma(LUAD)amino acid metabolism remains largely unknown.In this study,we found that UPF1 was significantly correlated with a portion of amino acid metabolic pathways in LUAD by integrating bioinformatics and metabolomics.We further confirmed that UPF1 knockdown inhibited activating transcription factor 4(ATF4)and Ser51 phosphorylation of eukaryotic translation initiation factor 2α(eIF2α),the core proteins in amino acid metabolism reprogramming.In addition,UPF1 promotes cell proliferation by increasing the amino-acid levels of LUAD cells,which depends on the function of ATF4.Clinically,UPF1 mRNA expression is abnormal in LUAD tissues,and higher expression of UPF1 and ATF4 was significantly correlated with poor overall survival(OS)in LUAD patients.Our findings reveal that UPF1 is a potential regulator of tumor-associated amino acid metabolism and may be a therapeutic target for LUAD.展开更多
文摘本研究通过编辑识别RNA结合蛋白超靶标(hyper targets of RNA-binding proteins identified by editing,HyperTRIBE)技术识别了HEK293细胞中上游移码蛋白1(up-frameshift protein 1,UPF1)的靶基因,并结合UPF1敲低数据研究其关键靶基因的生物学功能。实验设计采用三组平行质粒转染体系:实验组转染UPF1-ADAR2cd融合表达质粒、对照组转染mcherry-ADAR2cd质粒、背景组转染GFP空载体质粒。转染48 h后收集细胞进行转录组测序,通过HyperTRIBE识别HEK293细胞中UPF1的靶基因,并结合starBase数据库和UPF1敲低数据筛选UPF1关键靶基因。应用基因本体学、京都基因和基因组百科全书分析关键靶基因生物学功能,并使用相互作用基因检索工具绘制靶基因之间的蛋白质相互作用网络。最终,HyperTRIBE成功识别出1606个HEK293细胞中的UPF1靶基因,结合starBase数据库和UPF1敲低数据得到71个关键靶基因。这些靶基因在mRNA监测、细胞内囊泡转运、信号通路调控以及细胞器功能维持等多个生物学过程中发挥重要作用。研究结果证明了HyperTRIBE在识别RNA结合蛋白靶基因方面的有效性和准确性,为UPF1在其他细胞类型中的研究以及其他RNA结合蛋白的功能研究奠定了基础。
基金supported by the National Natural Science Foundation of China(Nos.81803886,81774078,and 21907093)。
文摘Up-frameshift 1(UPF1),as the most critical factor in nonsense-mediated messenger RNA(mRNA)decay(NMD),regulates tumor-associated molecular pathways in many cancers.However,the role of UPF1 in lung adenocarcinoma(LUAD)amino acid metabolism remains largely unknown.In this study,we found that UPF1 was significantly correlated with a portion of amino acid metabolic pathways in LUAD by integrating bioinformatics and metabolomics.We further confirmed that UPF1 knockdown inhibited activating transcription factor 4(ATF4)and Ser51 phosphorylation of eukaryotic translation initiation factor 2α(eIF2α),the core proteins in amino acid metabolism reprogramming.In addition,UPF1 promotes cell proliferation by increasing the amino-acid levels of LUAD cells,which depends on the function of ATF4.Clinically,UPF1 mRNA expression is abnormal in LUAD tissues,and higher expression of UPF1 and ATF4 was significantly correlated with poor overall survival(OS)in LUAD patients.Our findings reveal that UPF1 is a potential regulator of tumor-associated amino acid metabolism and may be a therapeutic target for LUAD.
