Graduate School of Science, University of Hyogo, Ako-gun, Hyogo, Japan.
Graduate School of Life Science, University of Hyogo, Ako-gun, Hyogo, Japan.
PLoS One. 2023 Dec 15;18(12):e0295659. doi: 10.1371/journal.pone.0295659. eCollection 2023.
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.
酿酒酵母在有葡萄糖的情况下使用高度糖酵解代谢，除了一些功能（如铁硫簇生物发生）外，适当地抑制线粒体功能。Puf3p 是 Pumillio 家族蛋白，通过使目标 mRNA 不稳定和/或抑制其翻译，在调节线粒体活性方面发挥着关键作用，尤其是在发酵过程中。Puf3p 优先结合核编码线粒体（nc-mitochondrial）mRNA 3'-UTR 中 8-nt 保守结合序列，导致在可发酵条件下对基因表达产生广泛影响。为了进一步探索 Puf3p 如何在转录后响应细胞生长条件调节 nc-mitochondrial mRNA，我们最初专注于在富含葡萄糖的环境中富集于单体的已知 nc-mitochondrial mRNA。我们出乎意料地发现，单体富集的 mRNA 之一 CAT5/COQ7 mRNA 通过其非典型的 Puf3p 结合序列与 Puf3p 直接相互作用，该序列通常被认为不是 Puf3p 结合位点。Western blot 分析表明，无论在可发酵或呼吸培养基中培养，Puf3p 均抑制 Cat5p 的翻译。体外结合实验证实 Puf3p 通过此非典型 Puf3p 结合位点与 CAT5 mRNA 直接相互作用。尽管非典型 Puf3p 结合位点的 cat5 突变体正常生长，但 Cat5p 的表达发生改变，表明 CAT5 mRNA 是 Puf3p 的真正靶标，并且有其他调节因子通过该序列发挥作用。与其他酵母 PUF 蛋白不同，Puf3p 通过使 mRNA 不稳定和抑制翻译来独特地调节 Cat5p，为 Puf3p 调控网络的未知部分提供了新的认识。鉴于人类 COQ7 的病理性变异导致 CoQ10 缺乏，并且酵母 cat5Δ可以被 hCOQ7 互补，我们的发现也可能为 COQ7 相关疾病的临床方面提供一些见解。
