Bremer Kirsten A, Stevens Audrey, Schoenberg Daniel R
Department of Molecular and Cellular Biochemistry and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.
RNA. 2003 Sep;9(9):1157-67. doi: 10.1261/rna.5720303.
beta-globin mRNA bearing a nonsense codon is degraded in the cytoplasm of erythroid cells by endonuclease cleavage, preferentially at UG dinucleotides. An endonuclease activity in polysomes of MEL cells cleaved beta-globin and albumin mRNA in vitro at many of the same sites as PMR1, an mRNA endonuclease purified from Xenopus liver. Stable transfection of MEL cells expressing normal human beta-globin mRNA with a plasmid vector expressing the catalytically active form of PMR1 reduced the half-life of beta-globin mRNA from 12 to 1-2 h without altering GAPDH mRNA decay. The reduced stability of beta-globin mRNA in these cells was accompanied by an increase in the production of mRNA decay products corresponding to those seen in the degradation of nonsense-containing beta-globin mRNA. Therefore, beta-globin mRNA is cleaved in vivo by an endonuclease with properties similar to PMR1. Inhibiting translation with cycloheximide stabilized nonsense-containing beta-globin mRNA, resulting in a fivefold increase in its steady-state level. Taken together, our results indicate that the surveillance of nonsense-containing beta-globin mRNA in erythroid cells is a cytoplasmic process that functions on translating mRNA, and endonucleolytic cleavage constitutes one step in the process of beta-globin mRNA decay.
携带无义密码子的β-珠蛋白mRNA在红细胞细胞质中通过核酸内切酶切割而降解，优先在UG二核苷酸处切割。MEL细胞多核糖体中的核酸内切酶活性在体外可在许多与从非洲爪蟾肝脏纯化的mRNA核酸内切酶PMR1相同的位点切割β-珠蛋白和白蛋白mRNA。用表达PMR1催化活性形式的质粒载体稳定转染表达正常人β-珠蛋白mRNA的MEL细胞，可使β-珠蛋白mRNA的半衰期从12小时缩短至1 - 2小时，而不改变甘油醛-3-磷酸脱氢酶（GAPDH）mRNA的降解。这些细胞中β-珠蛋白mRNA稳定性的降低伴随着与含无义β-珠蛋白mRNA降解中所见相对应的mRNA降解产物产量的增加。因此，β-珠蛋白mRNA在体内被一种性质类似于PMR1的核酸内切酶切割。用环己酰亚胺抑制翻译可使含无义的β-珠蛋白mRNA稳定，导致其稳态水平增加五倍。综上所述，我们的结果表明，红细胞中对含无义β-珠蛋白mRNA的监测是一个在翻译mRNA上起作用的细胞质过程，核酸内切酶切割是β-珠蛋白mRNA降解过程中的一个步骤。
