High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.
High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.
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DOI：
10.1093/nar/gkx297
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发表时间：
2017-07-07
影响因子：
14.9
通讯作者：
Gorospe M
中科院分区：
文献类型：
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作者：
Panda AC;De S;Grammatikakis I;Munk R;Yang X;Piao Y;Dudekula DB;Abdelmohsen K;Gorospe M
高通量RNA测序方法与专门的生物信息学分析相结合，最近发现了成千上万种独特的环状RNA，但它们的完整序列、起源基因和功能在很大程度上是未知的。由于环状rna缺乏自由末端，因此相对稳定，它们与microRNAs （miRNAs）和rna结合蛋白（rbp）的结合可以影响基因表达程序。虽然外核糖核酸酶处理被广泛用于降解线性RNA和富集RNA样品中的环状RNA，但它并不能有效地消除所有的线性RNA。在这里，我们描述了一种分离高纯度circRNA群体的新方法，包括RNase R处理，然后是聚腺酰化和聚(a)+ RNA耗竭（RPAD），这将线性RNA去除到接近完成。利用RPAD对人宫颈癌HeLa细胞和小鼠C2C12成肌细胞制备的RNA进行高通量测序，得出了两个令人惊讶的发现：(i)许多外显子环状RNA （EcircRNA）同种异构体具有相同的后剪接序列，但具有不同的体大小和序列；（ii）细胞中表达了数千种新的内含子环状RNA （icircrna）。总之，使用RPAD方法分离高纯度circRNA可以对circRNA类型和序列组成进行定量和定性分析，为阐明circRNA的功能铺平了道路。
High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.