裂殖酵母线粒体转录因子Mtf1 RNA甲基转移酶活性的探索

Mitochondrion has its own circular genome, whose transcription is conducted by mitochondrial RNA polymerase and its transcription factors. In yeast, apart from sequences similarity, the crystal structure of mitochondrial transcription factor Mtf1 resembles that of RNA methyltransferases. However biochemical proofs of this activity is still lacking. In this proposal, RNAs that bind to Mtf1 are captured by TAP co-immunoprecipitation, whose sequences are obtained by cDNA library construction and sequencing. Based on the sequence information, the RNAs methylated in vivo are verified by reverse transcription combined with DNA fragment scanning. The RNA methyltransferase activity of Mtf1 is also tested in vitro, thus confirming the RNA methyltransferase activity of Mtf1. Mtf1 point mutant strain that lack RNA methyltransferase activity is constructed. The biological role of the RNA methyltransferase activity of Mtf1 would be inferred from the morphological, physiological, biochemistry indexes of this mutant strain. The completion of this proposal offers new insight into the mitochondrial transcriptional regulation research, and also provides new approach for research on the mitochondrial related diseases.

线粒体具有独立的基因组，其转录由线粒体RNA聚合酶及其转录因子完成。酵母的线粒体转录因子Mtf1在蛋白序列和结构上具有RNA甲基转移酶的特征，然而目前还缺乏相关的生化证据证明该活性。本项目拟通过TAP亲和免疫共沉淀法获取与Mtf1结合的RNA，然后将RNA逆转录并克隆建库并测序，来获取与Mtf1直接结合的RNA序列信息，以此信息为基础进行体内RNA的甲基化检测，以及体外Mtf1甲基转移酶活性的测定来确认Mtf1是否具有甲基转移酶活性；构建Mtf1缺失甲基转移酶活性的突变菌株，通过观察该菌株的形态、生理、生化指标来推测Mtf1的甲基转移酶功能的生物学功能。该项目的完成将为线粒体转录调控机制的研究提供新的视角，并为线粒体相关的多种疾病研究开辟新思路。