Hartmuth Klaus, Vornlocher Hans-Peter, Lührmann Reinhard
Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany.
Methods Mol Biol. 2004;257:47-64. doi: 10.1385/1-59259-750-5:047.
The ability to isolate native ribonucleoprotein (RNP) particles is of fundamental importance in the study of processes such as pre-messenger RNA (mRNA) processing and translation. We have developed an RNA affinity tag that allows the large-scale preparation of native spliceosomes in a solid-phase assembly scheme. A tobramycin-binding aptamer cotranscriptionally added to the 3' end of the pre-mRNA is used to bind the pre-mRNA to tobramycin immobilized on a matrix. Incubation of the pre-mRNA thus immobilized allows the assembly of spliceosomes, which can be released from the matrix under native conditions by competition with tobramycin. Further density-gradient centrifugation affords highly purified spliceosomes suitable for the characterization of associated proteins by mass spectrometry as well as for studies using biochemical and biophysical methods. Although the method was developed for the preparation of spliceosomes, it is likewise applicable to the preparation of other RNP particles.
分离天然核糖核蛋白（RNP）颗粒的能力在诸如前体信使RNA（mRNA）加工和翻译等过程的研究中至关重要。我们开发了一种RNA亲和标签，可在固相组装方案中大规模制备天然剪接体。共转录添加到前体mRNA 3'末端的妥布霉素结合适体用于将前体mRNA与固定在基质上的妥布霉素结合。对如此固定的前体mRNA进行孵育可使剪接体组装，通过与妥布霉素竞争，可在天然条件下将其从基质中释放出来。进一步的密度梯度离心可提供高度纯化的剪接体，适用于通过质谱对相关蛋白质进行表征以及使用生化和生物物理方法进行研究。尽管该方法是为制备剪接体而开发的，但同样适用于制备其他RNP颗粒。
