Pluk H, van Eenennaam H, Rutjes S A, Pruijn G J, van Venrooij W J
Department of Biochemistry, University of Nijmegen, The Netherlands.
RNA. 1999 Apr;5(4):512-24. doi: 10.1017/s1355838299982079.
The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 5' end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1-86 and 116-176 of the MRP RNA to associate with the RNase MRP complex via protein-protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20- and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86-176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus- (VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38.
真核生物的核仁含有大量小RNA分子，这些小RNA分子以小核仁核糖核蛋白复合体（snoRNPs）的形式参与前体rRNA的加工和修饰。已被证明具有酶活性的一种snoRNP是核糖核酸酶MRP。核糖核酸酶MRP是一种参与5.8S rRNA 5'端形成的内切核糖核酸酶。在本研究中，对hPop1蛋白与核糖核酸酶MRP复合体的关联进行了研究。hPop1蛋白似乎不直接与RNA成分结合，而是需要MRP RNA的第1 - 86位核苷酸和116 - 176位核苷酸，通过蛋白质 - 蛋白质相互作用与核糖核酸酶MRP复合体结合。紫外线交联后进行核糖核酸酶处理，并用抗Th/To抗体进行免疫沉淀，结果显示有三种分子量约为20 kDa、25 kDa和40 kDa的人类蛋白质可与核糖核酸酶MRP复合体结合。20 kDa和25 kDa的蛋白质似乎与MRP RNA的茎环I结合，而40 kDa的蛋白质需要MRP RNA的中央部分（第86 - 176位核苷酸）才能与核糖核酸酶MRP复合体结合。此外，我们还表明人类核糖核酸酶P蛋白Rpp30和Rpp38也与核糖核酸酶MRP复合体相关联。在HeLa细胞中表达这些蛋白的水泡性口炎病毒（VSV）标记版本，随后进行抗VSV免疫沉淀，结果导致核糖核酸酶P和核糖核酸酶MRP复合体共沉淀。此外，紫外线交联后进行抗Th/To和抗Rpp38免疫沉淀表明，我们在紫外线交联中检测到的40 kDa蛋白质可能与Rpp38相同。
