Nuclear RNA processing requires dynamic and intricately regulated machinery composed of multiple enzymes and their cofactors.In this review,we summarize recent experiments using Saccharomyces cerevisiae as a model sys...Nuclear RNA processing requires dynamic and intricately regulated machinery composed of multiple enzymes and their cofactors.In this review,we summarize recent experiments using Saccharomyces cerevisiae as a model system that have yielded important insights regarding the conversion of pre-RNAs to functional RNAs,and the elimination of aberrant RNAs and unneeded intermediates from the nuclear RNA pool.Much progress has been made recently in describing the 3D structure of many elements of the nuclear degradation machinery and its cofactors.Similarly,the regulatory mechanisms that govern RNA processing are gradually coming into focus.Such advances invariably generate many new questions,which we highlight in this review.展开更多
目的构建hnRNP K特异性siRNA真核表达载体,体外观察对hnRNP K基因的沉默作用。方法采用基因克隆技术,将合成的特异性hnRNP K RNA干扰寡核苷酸序列插入真核表达载体pSUPER,构建hnRNP K siRNA真核表达载体。采用Lipofect AMINE2000将pSUPE...目的构建hnRNP K特异性siRNA真核表达载体,体外观察对hnRNP K基因的沉默作用。方法采用基因克隆技术,将合成的特异性hnRNP K RNA干扰寡核苷酸序列插入真核表达载体pSUPER,构建hnRNP K siRNA真核表达载体。采用Lipofect AMINE2000将pSUPER空载体和3个重组质粒(pSUPER/hnRNP K siRNAa,pSUPER/hnRNP K siRNAc和pSUPER/siRNAn)分别导入A549肺癌细胞(a、c、n分别代表hnRNP K编码序列中的A链和C链,以及无意义的对照序列Non链)。24 h后用RT-PCR和Western blot技术检测各实验组肺癌细胞内hnRNP K mRNA及蛋白水平的表达情况。结果成功构建了hnRNP K siRNA真核表达载体。转染hnRNP K siRNAa、hnRNP K siRNAc的肺癌细胞24 h后hnRNP K mRNA相对表达量分别为0.24±0.53和0.28±0.57,较对照组显著降低(P均<0.01);hnRNP K蛋白灰度值分别为0.23±0.11和0.28±0.09,较对照组显著降低(P均<0.05)。结论构建的RNA干扰真核表达载体能明显干扰A549细胞hnRNP K mRNA及蛋白的表达,为进一步研究hnRNP K基因的功能并应用于肺癌的治疗研究奠定了基础。展开更多
基金Supported by The Marlene and Stewart Greenebaum Cancer Center (Toth EA)
文摘Nuclear RNA processing requires dynamic and intricately regulated machinery composed of multiple enzymes and their cofactors.In this review,we summarize recent experiments using Saccharomyces cerevisiae as a model system that have yielded important insights regarding the conversion of pre-RNAs to functional RNAs,and the elimination of aberrant RNAs and unneeded intermediates from the nuclear RNA pool.Much progress has been made recently in describing the 3D structure of many elements of the nuclear degradation machinery and its cofactors.Similarly,the regulatory mechanisms that govern RNA processing are gradually coming into focus.Such advances invariably generate many new questions,which we highlight in this review.
文摘目的构建hnRNP K特异性siRNA真核表达载体,体外观察对hnRNP K基因的沉默作用。方法采用基因克隆技术,将合成的特异性hnRNP K RNA干扰寡核苷酸序列插入真核表达载体pSUPER,构建hnRNP K siRNA真核表达载体。采用Lipofect AMINE2000将pSUPER空载体和3个重组质粒(pSUPER/hnRNP K siRNAa,pSUPER/hnRNP K siRNAc和pSUPER/siRNAn)分别导入A549肺癌细胞(a、c、n分别代表hnRNP K编码序列中的A链和C链,以及无意义的对照序列Non链)。24 h后用RT-PCR和Western blot技术检测各实验组肺癌细胞内hnRNP K mRNA及蛋白水平的表达情况。结果成功构建了hnRNP K siRNA真核表达载体。转染hnRNP K siRNAa、hnRNP K siRNAc的肺癌细胞24 h后hnRNP K mRNA相对表达量分别为0.24±0.53和0.28±0.57,较对照组显著降低(P均<0.01);hnRNP K蛋白灰度值分别为0.23±0.11和0.28±0.09,较对照组显著降低(P均<0.05)。结论构建的RNA干扰真核表达载体能明显干扰A549细胞hnRNP K mRNA及蛋白的表达,为进一步研究hnRNP K基因的功能并应用于肺癌的治疗研究奠定了基础。
