Ford L P, Wilusz J
Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
Methods. 1999 Jan;17(1):21-7. doi: 10.1006/meth.1998.0703.
The pathways and machinery involved in the regulated turnover of mRNAs in mammalian cells are largely unknown. We have developed an in vitro system using HeLa cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates that faithfully reproduces in vivo aspects of regulated mRNA turnover. RNA substrates for use in the system that contain a poly(A) tail precisely at their 3' end can be readily prepared using a ligation-polymerase chain reaction approach. The system also uses standard cytoplasmic S100 extracts that are activated through the sequestration of poly(A)-binding proteins by the addition of cold poly(A) RNA. On incubation in the system, the poly(A) tail is removed from RNA substrates by a sequence-specific deadenylase activity and the body of the transcript is ultimately degraded in the system with no apparent intermediates by an ATP-dependent ribonulceolytic activity. AU-rich destability elements can regulate the rates of both deadenylation and degradation in the system. This in vitro system, therefore, should allow the elucidation of pathways of mRNA turnover, identification of the cellular factors involved, and insights into the mechanisms that regulate the half-life of a mRNA.
哺乳动物细胞中参与mRNA调控周转的途径和机制在很大程度上尚不清楚。我们开发了一种体外系统，该系统使用HeLa细胞质S100提取物和外源聚腺苷酸化RNA底物，能如实地再现体内调控mRNA周转的各个方面。使用连接-聚合酶链反应方法可以轻松制备用于该系统的RNA底物，这些底物在其3'端精确含有一个聚（A）尾。该系统还使用标准的细胞质S100提取物，通过添加冷聚（A）RNA来隔离聚（A）结合蛋白从而激活提取物。在该系统中孵育时，聚（A）尾通过序列特异性去腺苷酸酶活性从RNA底物上被去除，转录本的主体最终在该系统中通过依赖ATP的核糖核酸分解活性被降解，且无明显中间体。富含AU的不稳定元件可以调节该系统中去腺苷酸化和降解的速率。因此，这个体外系统应该能够阐明mRNA周转的途径，鉴定其中涉及的细胞因子，并深入了解调节mRNA半衰期的机制。
