Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [RNA-seq]. Homo sapiens

Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. Overall design: HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally.

可变mRNA剪接（alternative mRNA splicing）是基因调控与转录组多样性的重要机制之一。尽管该现象广泛存在，但剪接机器的调控机制与作用特异性仍仅部分得以阐明。由ADAR酶（ADAR enzymes）介导的前体mRNA（pre-mRNA）A-to-I RNA编辑（adenosine-to-inosine (A-to-I) RNA editing），已在多项研究案例中被证实与剪接调控相关。本研究采用生物信息学方法、RNA测序（RNA-seq）以及ADAR敲低细胞的外显子特异性微阵列，在全局层面探究了ADAR及其A-to-I RNA编辑活性对可变mRNA剪接的影响。尽管A-to-I RNA编辑极少靶向经典剪接受体、供体与分支位点，但研究发现其可作用于外显子内的剪接调控元件（splicing regulatory elements, SREs）。与组成型外显子相比，盒式外显子显著富集A-to-I RNA编辑位点。RNA-seq与外显子特异性微阵列分析显示，在肝癌与髓系白血病细胞系中敲低ADAR，会引发全局基因表达改变，且两个细胞系中均有数百个基因的剪接模式发生变化。这类全局剪接模式的改变无法仅通过推定的编辑位点来解释。剪接模式发生显著改变的基因，通常参与RNA加工与剪接活动。对近期公开的胶质母细胞瘤细胞系RNA-seq数据的分析也得到了相似结果。本全局分析表明，ADAR在剪接调控中发挥关键作用。尽管剪接基序确实存在直接编辑现象，但我们认为这并非ADAR介导的可变剪接调控的主要机制。相反，该调控是通过调节剪接机器中的反式作用因子（trans-acting factors）实现的。整体实验设计：将靶向特异性敲低ADAR表达的siRNA质粒稳定转染至HepG2与K562细胞系（ADAR KD），以此全局探究ADAR对可变剪接的调控作用。