Iwawaki Initiative Research Unit, Advanced Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Nucleic Acids Res. 2010 Oct;38(18):6265-73. doi: 10.1093/nar/gkq452. Epub 2010 May 27.
IRE1α is an endoplasmic reticulum (ER)-located transmembrane RNase that plays a central role in the ER stress response. Upon ER stress, IRE1α is activated and cleaves specific exon-intron sites in the mRNA encoding the transcription factor X-box-binding protein 1 (XBP1). In addition, previous studies allow us to predict that IRE1α targets several RNAs other than the XBP1. In fact, we have identified CD59 mRNA as a cleavage target of IRE1α. However, it is not yet clear how IRE1α recognizes and cleaves target RNAs. To address this question, we devised a unique method that combines an in vitro cleavage assay with an exon microarray analysis, and performed genome-wide screening for IRE1α cleavage targets. We identified 13 novel mRNAs as candidate IRE1α cleavage targets. Moreover, an analysis of the novel cleavage sites revealed a consensus sequence (CUGCAG) which, when accompanied by a stem-loop structure, is essential for IRE1α-mediated cleavage. The sequence and structure were also conserved in the known IRE1α cleavage targets, CD59 and XBP1. These findings provide the important clue to understanding the molecular mechanisms by which IRE1α recognizes and cleaves target RNAs.
IRE1α 是一种内质网 (ER) 定位的跨膜 RNA 酶，在 ER 应激反应中发挥核心作用。在 ER 应激时，IRE1α 被激活，并在编码转录因子 X 盒结合蛋白 1（XBP1）的 mRNA 中切割特定的外显子-内含子位点。此外，先前的研究使我们能够预测 IRE1α 除了 XBP1 之外还靶向几种 RNA。事实上，我们已经确定 CD59 mRNA 是 IRE1α 的切割靶标。然而，IRE1α 如何识别和切割靶标 RNA 尚不清楚。为了解决这个问题，我们设计了一种独特的方法，将体外切割测定法与外显子微阵列分析相结合，并进行了 IRE1α 切割靶标的全基因组筛选。我们鉴定了 13 个新的 mRNA 作为 IRE1α 切割靶标的候选物。此外，对新的切割位点的分析揭示了一个保守序列（CUGCAG），当与茎环结构一起存在时，对于 IRE1α 介导的切割是必需的。该序列和结构在已知的 IRE1α 切割靶标 CD59 和 XBP1 中也是保守的。这些发现为理解 IRE1α 识别和切割靶标 RNA 的分子机制提供了重要线索。
