Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer 52621, Ramat Gan, Israel.
RNA. 2013 May;19(5):591-604. doi: 10.1261/rna.038042.112. Epub 2013 Mar 8.
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.
选择性剪接是基因调控和转录组多样性的主要机制。尽管这种现象的程度很大，但剪接机制的调节和特异性仅部分得到理解。ADAR 酶对前体 mRNA 的腺苷到肌苷（A-to-I）RNA 编辑已在几种情况下与剪接调节相关。在这里，我们使用生物信息学方法、ADAR 敲低细胞的 RNA-seq 和外显子特异性微阵列，全面研究了 ADAR 及其 A-to-I RNA 编辑活性如何影响选择性 mRNA 剪接。尽管 A-to-I RNA 编辑很少靶向典型的剪接接受体、供体和分支位点，但它被发现影响外显子内的剪接调节元件（SRE）。与组成型外显子相比，盒式外显子被发现含有显著更多的 A-to-I RNA 编辑位点。RNA-seq 和外显子特异性微阵列显示，肝癌和髓样白血病细胞系中 ADAR 的敲低导致基因表达的全局变化，数百个基因在两种细胞系中改变了它们的剪接模式。这种剪接模式的全局变化不能仅用潜在的编辑位点来解释。显示剪接模式显著变化的基因通常涉及 RNA 处理和剪接活性。对最近发表的胶质母细胞瘤细胞系的 RNA-seq 数据进行分析显示了类似的结果。我们的全局分析表明，ADAR 在剪接调节中发挥主要作用。尽管确实发生了对剪接基序的直接编辑，但我们认为这不太可能是 ADAR 介导的选择性剪接调节的主要机制。相反，这种调节是通过调节参与剪接机制的反式作用因子来实现的。
