RNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation. However, the functions of RBPs in plants remain poorly understood. Maize kernel mutant dek42 has small defective kernels and ...RNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation. However, the functions of RBPs in plants remain poorly understood. Maize kernel mutant dek42 has small defective kernels and lethal seedlings. Dek42 was cloned by Mutator tag isolation and further confirmed by an independent mutant allele and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 materials. Dek42 encodes an RRM_RBM48 type RNA-binding protein that localizes to the nucleus. Dek42 is constitutively expressed in various maize tissues. The dek42 mutation caused a significant reduction in the accumulation of DEK42 protein in mutant kernels. RNA-seq analysis showed that the dek42 mutation significantly disturbed the expression of thousands of genes during maize kernel development. Sequence analysis also showed that the dek42 mutation significantly changed alternative splicing in expressed genes, which were especially enriched for the U12-type intron-retained type. Yeast two-hybrid screening identified SF3a1 as a DEK42-interacting protein. DEK42 also interacts with the spliceosome component U1-70K. These results suggested that DEK42 participates in the regulation of pre-messenger RNA splicing through its interaction with other spliceosome components. This study showed the function of a newly identified RBP and provided insights into alternative splicing regulation during maize kernel development.展开更多
目的筛选肺癌转移相关蛋白1(lung cancer metastasis related protein 1,LCMR1)的相互作用蛋白并进行验证。方法以人肺癌95D细胞RNA作为模板,构建95D细胞c DNA文库。将LCMR1质粒及人肺癌95D细胞c DNA文库共转化酵母菌AH109,初步筛选LCMR...目的筛选肺癌转移相关蛋白1(lung cancer metastasis related protein 1,LCMR1)的相互作用蛋白并进行验证。方法以人肺癌95D细胞RNA作为模板,构建95D细胞c DNA文库。将LCMR1质粒及人肺癌95D细胞c DNA文库共转化酵母菌AH109,初步筛选LCMR1相互作用蛋白。应用融合蛋白沉降技术和免疫共沉淀技术对酵母双杂交结果进行验证,确定蛋白质相互作用。结果成功构建了人肺癌95D细胞的c DNA文库。应用酵母双杂交技术筛选出6种LCMR1相互作用蛋白,选取其中DEK原癌基因进行验证。使用融合蛋白沉降技术及免疫共沉淀技术证实,LCMR1蛋白与DEK蛋白在体外及细胞内均可以特异结合,特异结合发生在DEK蛋白N端功能区。结论 LCMR1与DEK为相互作用蛋白,DEK蛋白N端功能区与细胞凋亡密切相关,为LCMR1基因功能研究提供线索和相应分子基础。展开更多
基金supported by the National Key Research and Development Program of China (2016YFD0101003)the National Natural Science Foundation of China (91635303 and 31425019)
文摘RNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation. However, the functions of RBPs in plants remain poorly understood. Maize kernel mutant dek42 has small defective kernels and lethal seedlings. Dek42 was cloned by Mutator tag isolation and further confirmed by an independent mutant allele and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 materials. Dek42 encodes an RRM_RBM48 type RNA-binding protein that localizes to the nucleus. Dek42 is constitutively expressed in various maize tissues. The dek42 mutation caused a significant reduction in the accumulation of DEK42 protein in mutant kernels. RNA-seq analysis showed that the dek42 mutation significantly disturbed the expression of thousands of genes during maize kernel development. Sequence analysis also showed that the dek42 mutation significantly changed alternative splicing in expressed genes, which were especially enriched for the U12-type intron-retained type. Yeast two-hybrid screening identified SF3a1 as a DEK42-interacting protein. DEK42 also interacts with the spliceosome component U1-70K. These results suggested that DEK42 participates in the regulation of pre-messenger RNA splicing through its interaction with other spliceosome components. This study showed the function of a newly identified RBP and provided insights into alternative splicing regulation during maize kernel development.
文摘目的筛选肺癌转移相关蛋白1(lung cancer metastasis related protein 1,LCMR1)的相互作用蛋白并进行验证。方法以人肺癌95D细胞RNA作为模板,构建95D细胞c DNA文库。将LCMR1质粒及人肺癌95D细胞c DNA文库共转化酵母菌AH109,初步筛选LCMR1相互作用蛋白。应用融合蛋白沉降技术和免疫共沉淀技术对酵母双杂交结果进行验证,确定蛋白质相互作用。结果成功构建了人肺癌95D细胞的c DNA文库。应用酵母双杂交技术筛选出6种LCMR1相互作用蛋白,选取其中DEK原癌基因进行验证。使用融合蛋白沉降技术及免疫共沉淀技术证实,LCMR1蛋白与DEK蛋白在体外及细胞内均可以特异结合,特异结合发生在DEK蛋白N端功能区。结论 LCMR1与DEK为相互作用蛋白,DEK蛋白N端功能区与细胞凋亡密切相关,为LCMR1基因功能研究提供线索和相应分子基础。
