Charteau Violette, Derksen Merel, Pruijn Ger J M
Department of Biomolecular Chemistry, Institute for Molecules and Materials (IMM), Radboud University, Nijmegen, The Netherlands.
Bio Protoc. 2023 Feb 20;13(4). doi: 10.21769/BioProtoc.4615.
Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for mitochondrial RNA processing (MRP) and RNase P are highly similar RNPs that play distinct roles at the cellular level; as a consequence, the specific isolation of either of these complexes is essential to study their biochemical function. Since their protein components are nearly identical, purification of these endoribonucleases using protein-centric methods is not feasible. Here, we describe a procedure employing an optimized high-affinity streptavidin-binding RNA aptamer, termed S1m, to purify RNase MRP free of RNase P. This report details all steps from the RNA tagging to the characterization of the purified material. We show that using the S1m tag allows efficient isolation of active RNase MRP.
RNA结合蛋白与RNA分子之间的相互作用是多种生物学过程的核心。因此，准确表征核糖核蛋白复合物（RNP）的组成至关重要。线粒体RNA加工核糖核酸酶（MRP）和核糖核酸酶P是高度相似的核糖核蛋白复合物，它们在细胞水平上发挥着不同的作用；因此，特异性分离这些复合物中的任何一种对于研究其生化功能至关重要。由于它们的蛋白质成分几乎相同，使用以蛋白质为中心的方法纯化这些核糖核酸内切酶是不可行的。在这里，我们描述了一种使用优化的高亲和力链霉亲和素结合RNA适体（称为S1m）来纯化不含核糖核酸酶P的核糖核酸酶MRP的方法。本报告详细介绍了从RNA标记到纯化材料表征的所有步骤。我们表明，使用S1m标签可以有效分离活性核糖核酸酶MRP。
