The discovery of reversible modifications in messenger RNA (mRNA) opens new research directions in RNA modification-mediated epigenetic regulation. Yeast is an extensively used model organism in molecular biology. Systematic investigation and profiling of modifications in yeast mRNA would promote our understanding of the physiological regulation mechanisms in yeast. However, due to the high abundance of ribosomal RNA (rRNA) and transfer RNA (tRNA) in total RNA, isolation of low abundance of mRNA frequently suffers from the contamination of rRNA and tRNA, which will lead to the false-positive determination and inaccurate quantification of modifications in mRNA. Therefore, obtaining high-purity mRNA is critical for precise determination and accurate quantification of modifications in mRNA, especially for studies that focus on discovering new ones. Herein, we proposed a successive orthogonal isolation method by combining polyT-based purification and agarose gel electrophoresis purification for extracting high-purity mRNA. With the extracted high-purity yeast mRNA, we systemically explored the modifications in yeast mRNA by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) analysis. The results showed that in addition to the previously reported eight kinds of modifications, two novel modifications of inosine (Ino) and 2′-O-methylinosine (Im) were identified to be prevalent in yeast mRNA. It is worth noting that Im was reported for the first time, to the best of our knowledge, to exist in living organisms in the three domains of life. Moreover, we observed that the levels of 10 kinds of modifications including Ino and Im in yeast mRNA exhibited dynamic change at different growth stages of yeast cells. Furthermore, Im in mRNA showed a significant decrease while in response to H₂O₂ treatment. These results indicated that the two newly identified modifications in yeast mRNA were involved in yeast cell growth and response to environmental stress. Taken together, we reported two new modifications of Ino and Im in yeast mRNA, which expends the diversity of RNA modifications in yeast and also suggests new regulators for modulating yeast physiological functions.

中文翻译：

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通过液相色谱-串联质谱分析鉴定酵母信使 RNA 中的肌苷和 2'-O-甲基肌苷修饰

信使 RNA (mRNA) 可逆修饰的发现为 RNA 修饰介导的表观遗传调控开辟了新的研究方向。酵母是分子生物学中广泛使用的模式生物。对酵母 mRNA 修饰的系统研究和分析将促进我们对酵母生理调节机制的理解。然而，由于总RNA中核糖体RNA（rRNA）和转移RNA（tRNA）的丰度较高，分离低丰度的mRNA时经常会出现rRNA和tRNA的污染，从而导致假阳性和不准确的测定。定量 mRNA 的修饰。因此，获得高纯度 mRNA 对于精确测定和准确定量 mRNA 修饰至关重要，尤其是对于专注于发现新修饰的研究。在此，我们提出了一种连续正交分离方法，将基于polyT的纯化和琼脂糖凝胶电泳纯化相结合，用于提取高纯度的mRNA。利用提取的高纯度酵母 mRNA，我们通过液相色谱-电喷雾电离-串联质谱 (LC-ESI-MS/MS) 分析系统地探索了酵母 mRNA 的修饰。结果表明，除此前报道的8种修饰外，还有肌苷（Ino）和2'- 我们通过液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）分析系统地探索了酵母mRNA的修饰。结果表明，除此前报道的8种修饰外，还有肌苷（Ino）和2'- 我们通过液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）分析系统地探索了酵母mRNA的修饰。结果表明，除此前报道的8种修饰外，还有肌苷（Ino）和2'-O-甲基肌苷 (Im) 被确定在酵母 mRNA 中普遍存在。值得注意的是，据我们所知，Im 首次被报道存在于生命的三个领域的生物体中。此外，我们观察到酵母mRNA中包括Ino和Im在内的10种修饰水平在酵母细胞的不同生长阶段表现出动态变化。此外，在响应 H ₂ O _{2时，mRNA 中的 Im 显着降低}治疗。这些结果表明，酵母mRNA中的两个新发现的修饰参与了酵母细胞的生长和对环境压力的反应。总之，我们报道了酵母 mRNA 中 Ino 和 Im 的两种新修饰，它们扩展了酵母中 RNA 修饰的多样性，并提出了调节酵母生理功能的新调节因子。