Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Methods Mol Biol. 2021;2192:103-115. doi: 10.1007/978-1-0716-0834-0_9.
RNA modifications are present in most cellular RNAs and are formed posttranscriptionally by enzymatic machineries that involve hundreds of enzymes and cofactors. RNA modifications impact the life cycle of the RNA, its stability, folding, cellular localization, as well as interactions with RNA and protein partners. RNA modifications are important for mitochondrial function and are required for proper processing and function of mitochondrial (mt) tRNA and rRNA. Underscoring their importance, several mitochondrial diseases are caused by defects in mt-RNA modifications, stemming from mutations in mtDNA at or near mt-RNA modification sites or in nuclear-encoded mt-RNA modifying enzymes. A highly abundant RNA modification, involved in mitochondrial physiology and pathology is pseudouridylation (Ψ), which is catalyzed by enzymes of the Pseudouridine Synthase (PUS) family. Although some Ψ sites in mt-rRNA and mt-tRNA have been identified, little is known about the functional role of these modifications. Furthermore, it is unknown which enzyme facilitates the modification of each site and it is likely that many yet undiscovered mt-RNA modifications exist, as is evidenced by recent work showing some Ψ sites on mRNA. Here, we present mito-Ψ-Seq, a high-throughput method for semiquantitative mapping of Ψ in mt-RNA.
RNA 修饰存在于大多数细胞 RNA 中，是由涉及数百种酶和辅助因子的酶促机制在后转录水平形成的。RNA 修饰影响 RNA 的生命周期、稳定性、折叠、细胞定位以及与 RNA 和蛋白质伴侣的相互作用。RNA 修饰对于线粒体功能很重要，并且是线粒体 (mt) tRNA 和 rRNA 正确加工和功能所必需的。几种线粒体疾病是由 mt-RNA 修饰缺陷引起的，这凸显了它们的重要性，这些缺陷源于 mtDNA 中 mt-RNA 修饰位点或核编码 mt-RNA 修饰酶的突变。一种高度丰富的 RNA 修饰，参与线粒体生理和病理学，是假尿嘧啶化 (Ψ)，它由假尿嘧啶合成酶 (PUS) 家族的酶催化。虽然已经鉴定出 mt-rRNA 和 mt-tRNA 中的一些 Ψ 位点，但这些修饰的功能作用知之甚少。此外，尚不清楚哪种酶促进每个位点的修饰，而且很可能存在许多尚未发现的 mt-RNA 修饰，最近的研究表明 mRNA 上存在一些 Ψ 位点就证明了这一点。在这里，我们提出了 mito-Ψ-Seq，这是一种用于 mt-RNA 中 Ψ 半定量作图的高通量方法。
