β-Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon

Previous work showed that human β-globin mRNAs harboring a premature termination codon are degraded in the erythroid tissues of mice to products that lack sequences from the mRNA 5′ end but contain a 5′ cap-like structure. Whether these decay products are the consequence of endonucleolytic or 5′-to-3′ exonucleolytic activity is unclear. We report that this β-globin mRNA decay pathway is recapitulated in cultured mouse erythroleukemia (MEL) cells and targets nonsense-free mRNA to a lesser extent than nonsense-containing mRNA. S1 nuclease mapping and primer extension demonstrated that 70–80% of decay product 5′ ends contain a UG dinucleotide. Detection of upstream counterparts of these decay products indicates that they are generated by endonucleolytic activity. Both crude and partially purified polysome extracts prepared from MEL cells contain an endonucleolytic activity that generates decay products comparable to those observed in vivo. These data suggest that an endonuclease with preference for UG dinucleotides is involved in the degradation of nonsense-containing and, to a lesser extent, nonsense-free human β-globin mRNAs in mouse erythroid cells.

既往研究表明，携带提前终止密码子（premature termination codon）的人类β-珠蛋白mRNA可在小鼠红系组织中被降解为缺失mRNA 5′端序列但保留5′帽类似结构（5′ cap-like structure）的产物。目前尚不清楚这些降解产物是内切核酸酶活性还是5′→3′外切核酸酶活性（5′-to-3′ exonucleolytic activity）介导的结果。本研究证实，该β-珠蛋白mRNA降解通路可在培养的小鼠红白血病（mouse erythroleukemia, MEL）细胞中重现，且其对携带无义突变的mRNA的靶向降解效率高于无无义突变的mRNA。S1核酸酶图谱分析（S1 nuclease mapping）与引物延伸实验（primer extension）结果显示，70%~80%的降解产物的5′端包含UG二核苷酸序列。对这些降解产物的上游对应序列的检测表明，它们由内切核酸酶活性介导产生。从MEL细胞中制备的粗提与部分纯化的多聚核糖体提取物，均含有可产生与体内观测结果一致的降解产物的内切核酸酶活性。上述数据提示，一种偏好识别UG二核苷酸的核酸内切酶，参与了小鼠红系细胞中携带无义突变的人类β-珠蛋白mRNA的降解，同时对无无义突变的该类mRNA也存在较弱的降解作用。