Garriga G, Lambowitz A M
Cell. 1984 Dec;39(3 Pt 2):631-41. doi: 10.1016/0092-8674(84)90470-7.
We have used Neurospora nuclear mutant cyt-18-1, which accumulates a number of unspliced mitochondrial precursor RNAs, to identify rapidly mitochondrial introns that are self-splicing in vitro. Incubation of deproteinized whole mitochondrial RNA from the mutant with 32P-GTP resulted in strong labeling of a 1.3 kb RNA, subsequently identified as cytochrome b (cob) intron 1, and weaker labeling of additional RNAs. Self-splicing of cob intron 1, including precise cleavage and ligation, was confirmed using an in vitro transcript synthesized from the SP6 promoter. The in vitro splicing reaction was shown to be analogous to that for the Tetrahymena nuclear rRNA intron. Since splicing of cob intron 1 is inhibited in a recessive nuclear mutant, we infer that this essentially RNA-catalyzed splicing reaction must be facilitated by a protein in vivo.
我们使用了 Neurospora 核突变体 cyt-18-1，该突变体积累了许多未剪接的线粒体前体 RNA，以快速鉴定出在体外可自我剪接的线粒体内含子。将来自该突变体的脱蛋白化全线粒体 RNA 与 32P-GTP 一起孵育，导致一条 1.3 kb RNA 强烈标记，随后鉴定为细胞色素 b（cob）内含子 1，其他 RNA 的标记较弱。使用从 SP6 启动子合成的体外转录本证实了 cob 内含子 1 的自我剪接，包括精确切割和连接。体外剪接反应显示与嗜热四膜虫核 rRNA 内含子的反应类似。由于 cob 内含子 1 的剪接在隐性核突变体中受到抑制，我们推断这种基本上由 RNA 催化的剪接反应在体内必定由一种蛋白质促进。
