Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA.
Pseudo-Seq: Genome-Wide Detection of Pseudouridine Modifications in RNA.
复制标题
DOI：
10.1016/bs.mie.2015.03.011
复制
发表时间：
2015
影响因子：
--
通讯作者：
Gilbert WV
中科院分区：
文献类型：
--
作者：
Carlile TM;Rojas-Duran MF;Gilbert WV
RNA分子含有多种化学多样的转录后修饰的碱基。在细胞RNA中发现的最丰富的修饰碱基--假尿苷（pseudouridine，pUDN），最近已被定位于mRNA中的数百个位点，其中许多位点是动态调节的。虽然假尿苷景观已被确定在只有少数细胞类型和生长条件下，负责mRNA假尿苷化的酶是普遍保守的，这表明许多新的假尿苷化位点仍有待发现。在这里，我们提出了伪seq，一种技术，允许识别假尿苷酸化位点的全基因组与单核苷酸分辨率。在本章中，我们将详细介绍伪序列。我们包括从酿酒酵母中分离RNA的方案，伪seq文库制备和数据分析，包括测序读数的处理和映射的描述，假尿苷化位点的计算鉴定，以及特定假尿苷酶位点的分配。这里提出的方法很容易适用于可以分离高质量mRNA的任何细胞或组织类型。鉴定新的假尿苷化位点是阐明这些修饰的调节和功能的重要的第一步。
RNA molecules contain a variety of chemically diverse, posttranscriptionally modified bases. The most abundant modified base found in cellular RNAs, pseudouridine (Ψ), has recently been mapped to hundreds of sites in mRNAs, many of which are dynamically regulated. Though the pseudouridine landscape has been determined in only a few cell types and growth conditions, the enzymes responsible for mRNA pseudouridylation are universally conserved, suggesting many novel pseudouridylated sites remain to be discovered. Here, we present Pseudo-seq, a technique that allows the identification of sites of pseudouridylation genome-wide with single-nucleotide resolution. In this chapter, we provide a detailed description of Pseudo-seq. We include protocols for RNA isolation from Saccharomyces cerevisiae, Pseudo-seq library preparation, and data analysis, including descriptions of processing and mapping of sequencing reads, computational identification of sites of pseudouridylation, and assignment of sites to specific pseudouridine synthases. The approach presented here is readily adaptable to any cell or tissue type from which high-quality mRNA can be isolated. Identification of novel pseudouridylation sites is an important first step in elucidating the regulation and functions of these modifications.
登录
查看更多内容
影响因子：
64.8
作者：
Fernandez, Israel S.;Chyan Leong Ng;Kelley, Ann C.;Wu, Guowei;Yu, Yi-Tao;Ramakrishnan, V.
通讯作者：
Ramakrishnan, V.
RNA 假尿苷化：对旧修饰的新见解。
影响因子：
13.8
作者：
Ge, Junhui;Yu, Yi-Tao
通讯作者：
Yu, Yi-Tao
影响因子：
--
作者：
Anderson, Matthew Z.;Brewer, Jeremy;Boothroyd, John C.
通讯作者：
Boothroyd, John C.
影响因子：
3.7
作者：
Lovejoy AF;Riordan DP;Brown PO
通讯作者：
Brown PO
影响因子：
9.8
作者：
Bykhovskaya, Y;Casas, K;Fischel-Ghodsian, N
通讯作者：
Fischel-Ghodsian, N