Guo Q B, Akins R A, Garriga G, Lambowitz A M
Department of Molecular Genetics, Ohio State University, Columbus 43210.
J Biol Chem. 1991 Jan 25;266(3):1809-19.
The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.
编码粗糙脉孢菌线粒体大核糖体RNA的基因含有一个2.3千碱基的I类内含子，该内含子在体外不能自我剪接。我们之前表明，该内含子在体内和体外的剪接依赖于粗糙脉孢菌的cyt-18蛋白，即线粒体酪氨酰-tRNA合成酶。在本研究中，我们对该内含子进行了进一步的结构分析，并构建了其突变衍生物，以确定剪接所需的特征或阻止其自我剪接的特征。先前的研究表明，该内含子在5'剪接位点附近含有一个大的发夹结构。通过绘制RNase III切割位点，我们将这个发夹结构鉴定为一个延伸的P2茎。我们通过删除426个氨基酸的内含子开放阅读框、大部分5'内含子发夹和所有的L8，构建了一个388个核苷酸的微型内含子。这个微型内含子与全长内含子表现出相同的蛋白质依赖性剪接，但仍然不能自我剪接。进一步的缺失，即去除所有的P2或全部或部分的P4、P6、P7或P9，会使剪接失活，这表明完整的I类内含子核心结构是必需的。加强P1、P10或P9.0配对并不能使微型内含子自我剪接。我们的研究结果表明，线粒体大核糖体RNA内含子不能自我剪接反映了在5'剪接位点进行切割所需的结构或活性的缺陷，这一缺陷要么存在于内含子核心本身，要么存在于核心与P1茎之间的相互作用中。
