Huang Hon-Ren, Chao Michael Y, Armstrong Barbara, Wang Yong, Lambowitz Alan M, Perlman Philip S
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, USA.
Mol Cell Biol. 2003 Dec;23(23):8809-19. doi: 10.1128/MCB.23.23.8809-8819.2003.
Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing.
酿酒酵母线粒体DNA II类内含子aI2的剪接依赖于内含子编码的62 kDa逆转录酶-成熟酶蛋白（p62）。在野生型菌株中，p62仍与切除的内含子套索RNA结合在核糖核蛋白（RNP）颗粒中，这些颗粒对于内含子归巢至关重要。对一种细菌II类内含子的研究表明，内含子结构域IV的DIVa亚结构是其成熟酶的高亲和力结合位点。在此，我们首先提供体外证据，将该结论扩展至aI2。然后，对aI2 DIVa突变菌株的实验表明，p62与DIVa的结合在体内对aI2剪接并非必需，但对归巢是必需的。由于DIVa突变菌株中的aI2剪接仍然依赖成熟酶，剪接必定依赖于其他RNA-蛋白质相互作用。在突变菌株中积累的p62具有逆转录酶活性，但在高盐和低盐浓度下的分级分离实验表明，它与内源性线粒体RNA的结合比野生型蛋白更弱，这种表型可能解释了归巢缺陷。用密切相关的内含子aI1的DIVa替换aI2的DIVa可改善体内剪接，但不能改善归巢，这表明DIVa有助于归巢所需的成熟酶-RNA相互作用的特异性。
