Probing Xist RNA Structure in Cells Using Targeted Structure-Seq.
Probing Xist RNA Structure in Cells Using Targeted Structure-Seq.
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DOI：
10.1371/journal.pgen.1005668
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发表时间：
2015-12
期刊：
影响因子：
4.5
通讯作者：
Simon MD
中科院分区：
文献类型：
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作者：
Fang R;Moss WN;Rutenberg-Schoenberg M;Simon MD
长链非编码RNA (lncRNA) Xist是哺乳动物细胞x染色体失活的主要调控因子。关于Xist和其他lncrna如何发挥作用的模型依赖于rna在细胞环境中形成的热力学稳定的二级和高阶结构。探测可接近的RNA碱基可以为构建RNA构象模型提供数据，从而深入了解RNA的功能、分子进化和模块化。为了研究细胞中Xist的结构，我们在RNA二级结构定位和建模的最新进展的基础上开发了靶向结构- seq，它结合了细胞中RNA结构的化学探测和靶向特异性大规模平行测序。通过富集来自感兴趣RNA的信号，Targeted Structure-Seq以相对较少的测序读数实现了对目标RNA的高覆盖率，从而提供了一种有针对性和可扩展的方法来分析细胞中的RNA构象。我们利用这种方法对全长Xist lncRNA进行探测，以建立Xist内功能元件的新模型，包括Xist 5 '端的重复A元件。该分析还发现了Xist中进化保守的新结构元件，包括对Xist功能很重要的靠近C重复序列的新元件。为了完成它们的工作，许多rna需要折叠成结构（通过碱基切割）。我们对一种特定的哺乳动物RNA的构象感兴趣，当它在细胞内时。Xist是一种非常大的非编码RNA (lncRNA)，长约17000 nt。Xist尤其重要，因为它是最早被发现的lncrna之一，可以关闭整个染色体上的基因。为了弄清楚Xist RNA是如何在小鼠细胞中折叠的，我们开发了一种新的方法，靶向结构-序列，来检查像Xist这样的大RNA的构象。利用计算机建模，我们确定了Xist中碱基配对成RNA双链的部分。我们还确定了Xist RNA的哪些部分可能是结构化的。这项工作为研究任何大RNA的二级结构提供了一种新的工具，并帮助我们了解Xist在细胞中发挥作用时的重要片段。
The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5’-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function. To do their jobs, many RNAs need to fold into structures (through base-paring). We were interested in the conformation of a specific mammalian RNA, Xist, when it is inside a cell. Xist is a very large non-coding RNA (lncRNA), that is >17,000 nt long. Xist is particularly important because it is one of the first lncRNAs to be discovered, and turns genes off across an entire chromosome. To figure out how Xist RNA is folded in mouse cells, we developed a new approach, Targeted Structure-Seq, to examine the conformation of large RNAs like Xist. Using computer modeling, we identified parts of Xist that are base paired into RNA duplexes. We also determined which parts of the Xist RNA are likely to be structured. This work provides a new tool for studying the secondary structure of any large RNA, and helps us understand what the important pieces of Xist look like while Xist does its work in the cell.