Wiryaman Timothy, Toor Navtej
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, United States.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, United States.
Methods. 2017 Aug 1;125:10-15. doi: 10.1016/j.ymeth.2017.06.020. Epub 2017 Jun 23.
Group II introns are self-splicing catalytic RNAs that are able to excise themselves from pre-mRNAs using a mechanism identical to that utilized by the spliceosome. Both structural and phylogenetic data support the hypothesis that group II introns and the spliceosome share a common ancestor. Structures of group II introns have given insight into the active site required for the catalysis of RNA splicing. This review outlines crucial aspects of the structure determination of group II introns such as sample preparation and data processing. Given that group II introns are large RNAs that must be synthesized through in vitro transcription, there are special considerations that must be taken into account in terms of purification and crystallization, as compared to the isolation of large intact ribonucleoprotein complexes such as the ribosome. We specifically focus on the methodology used to determine the structure of the eukaryotic group II intron lariat from the brown algae Pylaiella littoralis. The techniques described in this review can also be applied for the structure determination of other large RNAs.
II 类内含子是自我剪接的催化 RNA，能够利用与剪接体相同的机制从 pre-mRNA 中自我切除。结构和系统发育数据均支持 II 类内含子和剪接体有共同祖先的假说。II 类内含子的结构为深入了解 RNA 剪接催化所需的活性位点提供了线索。本综述概述了 II 类内含子结构测定的关键方面，如样品制备和数据处理。鉴于 II 类内含子是必须通过体外转录合成的大型 RNA，与分离核糖体等大型完整核糖核蛋白复合物相比，在纯化和结晶方面必须考虑一些特殊因素。我们特别关注用于确定褐藻小海带真核 II 类内含子套索结构的方法。本综述中描述的技术也可应用于其他大型 RNA 的结构测定。
