Mangus D A, Jacobson A
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122, USA.
Methods. 1999 Jan;17(1):28-37. doi: 10.1006/meth.1998.0704.
mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. In Saccharomyces cerevisiae, several trans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeast UPF1 protein to test whether such interactions are altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperature-sensitive eIF3 mutation to the nonpermissive temperature. These experiments, as well as others assaying the strength of factor association in high-salt sucrose gradients, lead us to conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to the 40S or 60S ribosomal subunits. Similar experimental approaches were used to determine whether mRNA decay initiates prior to mRNA release from polyribosomes. Using sucrose gradient fractionation and Northern blotting, we can detect the polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA decay commences while an mRNA is still being translated.
mRNA降解是一个多步骤过程，通常依赖于mRNA的活跃翻译以及翻译装置的组成部分。在酿酒酵母中，mRNA降解所需的几种反式作用因子与多核糖体相关联。我们利用酵母UPF1蛋白的蔗糖梯度沉降分析，探究了这些因子与多核糖体相互作用的特异性，以测试当多核糖体通过用EDTA处理、微球菌核酸酶消化或含有温度敏感型eIF3突变的细胞转移至非允许温度而被破坏时，这种相互作用是否会改变。这些实验以及其他在高盐蔗糖梯度中测定因子结合强度的实验，使我们得出结论：Upf1p紧密结合于最小的多核糖体，但不与40S或60S核糖体亚基结合。类似的实验方法被用于确定mRNA降解是否在mRNA从多核糖体释放之前启动。通过蔗糖梯度分级分离和Northern印迹法，我们能够检测到PGK1 mRNA降解中间体的多核糖体关联，并得出结论：mRNA降解在mRNA仍在翻译时就开始了。
