Investigation of RNA editing sites within bound regions of RNA-binding proteins

Studies in epitranscriptomics indicates that RNA is modified by a variety of enzymes. Among these RNA modifications, A-to-I RNA editing occurs frequently in the mammalian transcriptome. These RNA editing sites can be detected directly from RNA-seq data by examining nucleotide changes from adenosine (A) to guanine (G), which substitutes for inosine (I). However, a careful investigation of such nucleotide changes must be conducted to distinguish sequencing errors and genomic mutations from the genuine editing sites. Building upon our recent introduction of an easy-to-use bioinformatics tool, RNAEditor, to detect RNA editing events from RNA-seq data, we examined the extent by which RNA editing events affects the binding of RNA-binding proteins (RBP). Through employing bioinformatic techniques, we uncover that RNA editing sites occur frequently in RBP-bound regions. Moreover, the presence of RNA editing sites are more frequent when RNA editing islands are examined, which are regions in which RNA editing sites are present in clusters. When the binding of one RBP, HuR, was quantified experimentally, its binding was reduced upon silencing of the RNA editing enzyme ADAR compared to the control—suggesting that the presence of RNA editing islands influences HuR binding to its target regions. These data indicate RNA editing as an important mediator of RBP-RNA interactions—a mechanism which likely constitutes an additional mode of post-transcription gene regulation in biological systems. Overall design: The RNA editing enzyme, ADAR, was silenced in HEK-293 cells.

表观转录组学（epitranscriptomics）研究表明，RNA可被多种酶修饰。在这类RNA修饰中，A-to-I RNA编辑（A-to-I RNA editing）在哺乳动物转录组中频繁发生。此类RNA编辑位点可直接从RNA测序（RNA-seq）数据中检测得到，具体方式为比对腺苷（A）向鸟苷（G）的核苷酸变化——该变化对应肌苷（I）的替换。然而，需对这类核苷酸变化进行严谨分析，以将测序误差、基因组突变与真实编辑位点区分开来。
基于我们此前推出的一款可便捷从RNA-seq数据中检测RNA编辑事件的生物信息学工具RNAEditor，我们探究了RNA编辑事件对RNA结合蛋白（RNA-binding proteins, RBP）结合活性的影响程度。通过生物信息学分析手段，我们发现RNA编辑位点在RBP结合区域中频繁出现。此外，当分析RNA编辑岛（RNA editing islands，即RNA编辑位点成簇分布的区域）时，RNA编辑位点的出现频率更高。我们通过实验量化了其中一种RBP——HuR的结合活性，结果显示，与对照组相比，沉默RNA编辑酶ADAR后，HuR的结合活性有所下降——这表明RNA编辑岛的存在会影响HuR与其靶区域的结合。
上述数据表明，RNA编辑是RBP与RNA相互作用的重要介导因子，这一机制可能构成生物系统中转录后基因调控的另一重方式。
整体实验设计：在HEK-293细胞中沉默RNA编辑酶ADAR。