Goguel V, Delahodde A, Jacq C
Laboratoire de Génétique Moléculaire, CNRS URA 1302, Ecole Normale Supérieure, Paris, France.
Mol Cell Biol. 1992 Feb;12(2):696-705. doi: 10.1128/mcb.12.2.696-705.1992.
The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity.
内含子编码的蛋白质bI4 RNA成熟酶和aI4 DNA内切酶可以从其线粒体编码序列的工程形式在酵母细胞质中如实地表达。在这项工作中，我们研究了与两种同源内含子编码蛋白质相关的这两种活性之间的关系：细胞色素b基因的第四个内含子中编码的bI4 RNA成熟酶和细胞色素氧化酶亚基I编码基因的第四个内含子中编码的aI4 DNA内切酶（I-SceII）。利用酵母的高重组特性以及这两个基因之间的相似性，我们在体内构建了一个携带RNA成熟酶和DNA内切酶编码序列部分的杂交基因家族。在这些杂交基因上游存在编码线粒体靶向肽的序列，使我们能够在体内研究其翻译产物在线粒体内的特性。因此，我们可以分析重组蛋白在不同菌株中补充RNA成熟酶缺陷的能力。在重组体中发现了两种亲本内含子序列的许多组合。它们的结构和功能分析揭示了以下特征。（i）bI4 RNA成熟酶的N端一半可以完全被来自aI4 DNA内切酶的等效部分取代，而不会影响RNA成熟酶活性。相反，用来自aI4 DNA内切酶的等效部分替换bI4 RNA成熟酶的C端一半会导致非常弱的RNA成熟酶活性，表明该区域更具差异性并且与成熟酶活性相关。（ii）没有一种具有RNA成熟酶活性的杂交蛋白保留DNA内切酶活性，这表明后者需要aI4蛋白的完整性。这些观察结果很有趣，因为已知aI4 DNA内切酶在DNA水平上促进aI4内含子的传播，而其编码内含子剪接所需的bI4 RNA成熟酶也控制aI4内含子的剪接过程。我们提出了一种这些内含子蛋白进化的设想，该设想依赖于从DNA内切酶活性到RNA成熟酶活性的转变。
