tRNA Modifications as a Readout of S and Fe-S Metabolism.
tRNA Modifications as a Readout of S and Fe-S Metabolism.
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DOI：
10.1007/978-1-0716-1605-5_8
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Dos Santos PC
中科院分区：
文献类型：
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作者：
Edwards AM;Addo MA;Dos Santos PC
铁-硫（Fe-S）簇作为已知调节金属酶活性的核心辅基，充当生物铁和硫的运输载体，并参与几个交叉的代谢途径。这些簇的形成是由一类称为半胱氨酸脱硫酶的酶引发的，其主要功能是将硫从氨基酸L-半胱氨酸转运到参与Fe-S簇合成以及其他硫辅因子合成的各种硫转移蛋白。因此，硫和Fe-S簇代谢通过共享的酶中间体连接，并且其相关途径中的缺陷导致无数的多效性表型，这是难以剖析的。转录后修饰的转移RNA（tRNA）代表了一大类分析物，其合成通常需要硫转移酶和Fe-S酶的协调参与。因此，这些分子可用作细胞Fe和S状态的生物学相关读数。采用LC-MS技术的方法提供了一种有价值的实验工具，以确定生物样品中tRNA修饰的相对水平，从而评估依赖于Fe-S簇的遗传、营养和环境因素调节反应。在本文中，我们描述了一种用于提取RNA并通过LC-MS平台分析评估Fe-S依赖性和非依赖性tRNA修饰程度的稳健方法。
Iron-Sulfur (Fe-S) clusters function as core prosthetic groups known to modulate the activity of metalloenzymes, act as trafficking vehicles for biological iron and sulfur, and participate in several intersecting metabolic pathways. The formation of these clusters is initiated by a class of enzymes called cysteine desulfurases, whose primary function is to shuttle sulfur from the amino acid L-cysteine to a variety of sulfur transfer proteins involved in Fe-S cluster synthesis as well as in the synthesis of other thiocofactors. Thus, sulfur and Fe-S cluster metabolism are connected through shared enzyme intermediates, and defects in their associated pathways cause a myriad of pleiotropic phenotypes, which are difficult to dissect. Post-transcriptionally modified transfer RNA (tRNA) represents a large class of analytes whose synthesis often requires the coordinated participation of sulfur transfer and Fe-S enzymes. Therefore, these molecules can be used as biologically relevant readouts for cellular Fe and S status. Methods employing LC-MS technology provide a valuable experimental tool to determine the relative levels of tRNA modification in biological samples and, consequently, to assess genetic, nutritional, and environmental factors modulating reactions dependent on Fe-S clusters. Herein, we describe a robust method for extracting RNA and analytically evaluating the degree of Fe-S-dependent and -independent tRNA modifications via an LC-MS platform.