Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States.
Methods Enzymol. 2021;658:277-310. doi: 10.1016/bs.mie.2021.06.026. Epub 2021 Jul 30.
Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNAs. High-throughput pseudouridine profiling of eukaryotic mRNAs from cells has revealed novel sites of modification across the transcriptome. Pseudouridine affects RNA structure and RNA-protein interactions with the potential to influence many steps of mRNA metabolism and thereby affect gene expression. Identifying the mechanisms by which individual pseudouridines sites are modified by pseudouridine synthases (PUS) will facilitate studies on the molecular functions of Ψ. Multiple pseudouridine synthases are expressed in all organisms and might direct pseudouridylation of diverse cellular RNAs, but the RNA targets of many enzymes and their specificity determinants remain to be defined. We developed a high-throughput in vitro pseudouridylation assay followed by sequencing that allows validation of candidate sites identified in cells, assignment of sites as direct targets of PUS and interrogation of the RNA sequence and structural features that direct modification. We also implemented an analysis pipeline to assign Ψ sites from these data, including an updated approach to peak-calling that accounts for noisy signal from low-abundance transcripts.
假尿嘧啶核苷 (Ψ) 是细胞 RNA 中最丰富的修饰物之一。从细胞中高通量鉴定真核生物 mRNA 的假尿嘧啶核苷谱，揭示了转录组中修饰的新位点。假尿嘧啶核苷影响 RNA 结构和 RNA-蛋白质相互作用，有可能影响 mRNA 代谢的许多步骤，从而影响基因表达。确定单个假尿嘧啶核苷位点被假尿嘧啶核苷合成酶 (PUS) 修饰的机制，将有助于研究 Ψ 的分子功能。多种假尿嘧啶核苷合成酶在所有生物体中表达，并可能指导多种细胞 RNA 的假尿嘧啶化，但许多酶的 RNA 靶标及其特异性决定因素仍有待确定。我们开发了一种高通量的体外假尿嘧啶化测定法，随后进行测序，允许验证在细胞中鉴定出的候选位点，将位点指定为 PUS 的直接靶标，并研究指导修饰的 RNA 序列和结构特征。我们还实施了一个分析管道，从这些数据中分配 Ψ 位点，包括一种更新的峰调用方法，该方法考虑了低丰度转录本的嘈杂信号。
