Plasmids used in this study.

The Saccharomyces cerevisiae uses a highly glycolytic metabolism, if glucose is available, through appropriately suppressing mitochondrial functions except for some of them such as Fe/S cluster biogenesis. Puf3p, a Pumillio family protein, plays a pivotal role in modulating mitochondrial activity, especially during fermentation, by destabilizing its target mRNAs and/or by repressing their translation. Puf3p preferentially binds to 8-nt conserved binding sequences in the 3′-UTR of nuclear-encoded mitochondrial (nc-mitochondrial) mRNAs, leading to broad effects on gene expression under fermentable conditions. To further explore how Puf3p post-transcriptionally regulates nc-mitochondrial mRNAs in response to cell growth conditions, we initially focused on nc-mitochondrial mRNAs known to be enriched in monosomes in a glucose-rich environment. We unexpectedly found that one of the monosome-enriched mRNAs, CAT5/COQ7 mRNA, directly interacts with Puf3p through its non-canonical Puf3p binding sequence, which is generally less considered as a Puf3p binding site. Western blot analysis showed that Puf3p represses translation of Cat5p, regardless of culture in fermentable or respiratory medium. In vitro binding assay confirmed Puf3p’s direct interaction with CAT5 mRNA via this non-canonical Puf3p-binding site. Although cat5 mutants of the non-canonical Puf3p-binding site grow normally, Cat5p expression is altered, indicating that CAT5 mRNA is a bona fide Puf3p target with additional regulatory factors acting through this sequence. Unlike other yeast PUF proteins, Puf3p uniquely regulates Cat5p by destabilizing mRNA and repressing translation, shedding new light on an unknown part of the Puf3p regulatory network. Given that pathological variants of human COQ7 lead to CoQ10 deficiency and yeast cat5Δ can be complemented by hCOQ7, our findings may also offer some insights into clinical aspects of COQ7-related disorders.

酿酒酵母（Saccharomyces cerevisiae）拥有高度糖酵解型代谢模式：当葡萄糖可及时，其会特异性抑制线粒体的多数功能，仅保留铁硫簇生物发生（Fe/S cluster biogenesis）等少数关键过程。Puf3蛋白(Puf3p)属于Pumilio家族，在调控线粒体活性（尤其在发酵过程中）中发挥核心作用，其机制为通过降解靶标信使RNA（mRNA）或抑制其翻译来实现调控。Puf3p可优先结合核编码线粒体mRNA（nc-mitochondrial mRNAs）3'非翻译区（3′-UTR）内的8核苷酸保守结合序列，在可发酵培养条件下对基因表达产生广泛影响。为进一步解析Puf3p如何响应细胞生长条件、对核编码线粒体mRNA进行转录后调控，我们最初将研究焦点置于已知在富葡萄糖环境中富集于单核糖体（monosomes）的核编码线粒体mRNA。我们意外发现，其中一类单核糖体富集mRNA——CAT5/COQ7 mRNA——可通过其非典型Puf3p结合序列与Puf3p直接互作，这类序列通常未被认定为Puf3p的结合位点。蛋白质印迹（Western blot）分析结果显示，无论培养于发酵型还是呼吸型培养基，Puf3p均能抑制Cat5p的翻译。体外结合实验（in vitro binding assay）证实，Puf3p可通过该非典型Puf3p结合位点与CAT5 mRNA直接结合。尽管携带该非典型Puf3p结合位点突变的cat5菌株生长状态正常，但Cat5p的表达水平发生了显著改变，这表明CAT5 mRNA是真正的Puf3p靶标，且存在额外调控因子通过该序列发挥调控作用。与其他酵母PUF家族蛋白不同，Puf3p可同时通过介导mRNA降解与翻译抑制来独特调控Cat5p，这为Puf3p调控网络中尚未被阐明的部分提供了全新视角。鉴于人类COQ7的病理性变异会导致辅酶Q10（CoQ10）缺乏症，且酵母cat5Δ突变体可通过人源COQ7（hCOQ7）实现功能互补，本研究结果或可为COQ7相关疾病的临床研究提供有价值的参考见解。