The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3′-UTRs of yeast mitochondrial mRNAs

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5′ capping and 3′ polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3′-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3′-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.信使RNA（messenger RNA，mRNA）的稳定化是转录后基因调控中的关键步骤。在真核细胞的细胞核与细胞质中，该过程通常通过5'端加帽（5' capping）与3'端多聚腺苷酸化（3' polyadenylation）完成；细菌与细胞器中则存在额外的调控机制。酿酒酵母（Saccharomyces cerevisiae）的线粒体mRNA含有一类十二聚体序列元件（dodecamer sequence element），该元件可通过尚未阐明的机制赋予RNA稳定性并介导3'端加工。本研究分离得到结合该十二聚体元件的蛋白质，并鉴定其为Rmd9——一种此前已被证实可稳定酿酒酵母线粒体RNA的因子。实验证实，Rmd9在体内可与十二聚体元件周边的mRNA结合，且重组Rmd9在体外可特异性结合该元件。Rmd9与其十二聚体靶标结合的晶体结构显示，Rmd9属于五肽重复蛋白（pentatricopeptide repeat, PPR）家族，并采用了此前未被报道的特异性RNA识别模式。体外实验表明，Rmd9可保护RNA免受线粒体3'外切核糖核酸酶复合物mtEXO的降解，这提示Rmd9对十二聚体元件的识别与结合，赋予了酿酒酵母线粒体mRNA稳定性。