Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing.
Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing.
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DOI：
10.1016/bs.mie.2021.06.026
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发表时间：
2021
影响因子：
--
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假尿苷（Pseudouridine，PGD）是细胞RNA中最丰富的修饰之一。来自细胞的真核mRNA的高通量假尿苷分析揭示了转录组中的新修饰位点。假尿苷影响RNA结构和RNA-蛋白质相互作用，可能影响mRNA代谢的许多步骤，从而影响基因表达。确定假尿苷酶（PUS）修饰单个假尿苷位点的机制将有助于研究PUS的分子功能。多种假尿苷磷酸酶在所有生物体中表达，并可能指导不同细胞RNA的假尿苷化，但许多酶的RNA靶点及其特异性决定簇仍有待确定。我们开发了一种高通量的体外假尿苷化测定，然后进行测序，从而可以验证细胞中鉴定的候选位点，将位点分配为PUS的直接靶点，并询问指导修饰的RNA序列和结构特征。我们还实施了一个分析管道来从这些数据中分配重复位点，包括一种更新的峰值调用方法，该方法考虑了来自低丰度转录本的噪声信号。
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.
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影响因子：
7
作者：
Safra M;Nir R;Farouq D;Vainberg Slutskin I;Schwartz S
通讯作者：
Schwartz S
DOI：
10.1093/bioinformatics/btq033
发表时间：
2010-03-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Quinlan AR;Hall IM
通讯作者：
Hall IM
影响因子：
14.9
作者：
Jiang, HQ;Motorin, Y;Grosjean, H
通讯作者：
Grosjean, H
影响因子：
14.9
作者：
Bailey TL;Johnson J;Grant CE;Noble WS
通讯作者：
Noble WS
DOI：
10.1007/978-1-0716-1374-0_22
发表时间：
2021-01-01
期刊：
RNA MODIFICATIONS
影响因子：
--
作者：
Martinez, Nicole M.;Gilbert, Wendy V.
通讯作者：
Gilbert, Wendy V.