PPR蛋白是一类广泛存在于真核生物中的RNA结合蛋白，参与细胞器RNA的加工、编辑、剪切、稳定性及翻译等RNA代谢过程。我们前期研究发现，裂殖酵母PPR蛋白Ppr10和Mpa1形成复合物，并且这个复合物参与线粒体蛋白合成，但作用机制不清楚。本项目拟以生化手段、遗传手段及高通量RNA测序技术，进一步研究Ppr10-Mpa1复合物在线粒体翻译中的作用。首先，通过体外生化实验和功能互补实验，研究Ppr10和Mpa1相互作用的分子机制及其生物学意义。采用RNA凝胶迁移方法及光活性增强的核糖核苷交联和免疫共沉淀(PAR-CLIP)方法，确定Ppr10-Mpa1复合物识别线粒体mRNA的结合位点。最后，通过线粒体核糖体图谱分析和线粒体核糖体指纹分析，研究Ppr10-Mpa1复合物如何在线粒体蛋白合成中发挥作用。该课题的研究有助于解析PPR蛋白识别靶RNA的分子密码及认识线粒体蛋白合成的机制。

The pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are found in eukaryotes. PPR proteins participate in RNA metabolism including 5’-end processing of rRNA and tRNA, as well as editing，splicing，stability and translation of mRNA, and thus are essential to organellar gene expression. In our previous studies, we have found that Schizosaccharomyces pombe Ppr10 and a novel protein Mpa1 form a complex which is required for mitochondrial protein synthesis, but the underlying mechanism is unknown. In this project, we will use biochemical, genetic and high-throughput sequencing approaches to elucidate the mechanism by which the Ppr10-Mpa1 complex activates mitochondrial translation. First, we will use in vitro biochemical approaches and functional complementation assays to investigate the molecular mechanism and the biological significance of the interaction between Ppr10 and Mpa1. Next, we will use the RNA gel mobility shift assay and the PAR-CLIP approach to identify the RNA binding site (s) of the Ppr10-Mpa1 complex. Lastly, we will investigate the role of the Ppr10-Mpa1 complex in mitochondrial translation using mitoribosome profiling and mitoribosome footprinting. The results from this project should help understand the molecular code for how PPR proteins recognize their target RNAs and provide new insights into the mechanism of mitochondrial protein synthesis.