Lustig A J, Lin R J, Abelson J
Cell. 1986 Dec 26;47(6):953-63. doi: 10.1016/0092-8674(86)90810-x.
The yeast rna mutations (rna2-rna11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases (rna2, rna5, and rna11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.
酵母RNA突变(rna2 - rna11)是一组温度敏感型突变,在限制温度下会导致含内含子的mRNA前体积累。我们利用酵母体外剪接系统来研究RNA基因产物在mRNA剪接中的作用。我们测试了从突变型和野生型细胞中分离的提取物中体外mRNA剪接反应的热不稳定性。在该检测中,从九个rna突变体中的七个分离出的提取物表现出热不稳定性,而大多数野生型提取物在所使用的条件下是稳定的。我们还通过表明来自不同突变体的热灭活提取物的大多数组合相互互补,证明热失活通常会导致一种可交换成分的特异性丧失。在三种情况(rna2、rna5和rna11)下,通过回复、四分体和体外互补分析相结合的方法确定了体外缺陷与rna突变的联系。此外,每种热灭活提取物都能够被野生型剪接系统的至少一种组分互补。因此,许多RNA基因可能编码直接参与mRNA剪接且对其至关重要的产物。