Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
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DOI：
10.3390/genes10010026
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发表时间：
2019-01-01
期刊：
影响因子：
3.5
通讯作者：
Kellner, Stefanie
中科院分区：
文献类型：
--
作者：
Borland, Kayla;Diesend, Jan;Kellner, Stefanie
已经发现转录后RNA修饰存在于多种生物体和不同类型的RNA中。核苷修饰由于其在翻译保真度、酶识别、疾病进展和RNA稳定性中的已知作用而令人感兴趣。此外，修饰核苷的丰度基于生长阶段、外部应力或可能的其他尚未探索的因素而波动。随着修饰的不断变化，需要一种方法来确定多个核苷修饰的绝对量。在这里，我们报告代谢同位素标记，以产生同位素标记的细菌和酵母的内标。这些可用于26种不同修饰核苷的定量。我们详细解释了这些内标物是如何产生的，并展示了它们的质谱表征。我们应用我们的内部标准品，并量化从细菌和各种真核生物的转移RNA（tRNA）的修饰含量。我们可以证明内标物的来源对定量结果无影响。此外，我们使用我们的内标物来定量小鼠组织信使RNA（mRNA）中的修饰核苷，其中我们在肝脏和脑组织中发现了不同的修饰谱。
Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.