Chang T H, Clark M W, Lustig A J, Cusick M E, Abelson J
Division of Biology, California Institute of Technology, Pasadena 91125.
Mol Cell Biol. 1988 Jun;8(6):2379-93. doi: 10.1128/mcb.8.6.2379-2393.1988.
The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.
酵母RNA突变(rna2至rna10/11)是一组温度敏感型突变,在非允许温度下会导致前体mRNA积累。大多数酵母RNA基因产物参与体外mRNA剪接并对其至关重要,这表明它们编码剪接机制的组分。我们通过使用体外合成的RNA11蛋白来弥补rna11提取物的温度敏感缺陷,对这一推测进行了验证。在体外互补过程中,输入的RNA11蛋白与40S剪接体和一个30S复合物相关联,这表明RNA11蛋白确实是剪接体的一个组分。RNA11相关30S复合物的形成不需要任何外源RNA底物,这表明这个30S颗粒可能是一个参与剪接的预组装复合物。RNA11特异性抗体在体外抑制mRNA剪接,证实了RNA11蛋白在mRNA剪接中的关键作用。最后,使用抗RNA11抗体,我们将RNA11蛋白定位到酵母细胞核的周边。