Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
RNA. 2011 Nov;17(11):1973-82. doi: 10.1261/rna.2747411. Epub 2011 Sep 26.
X-chromosome inactivation (XCI) in female mammals depends on the noncoding RNA X inactivation specific transcript (Xist). The mechanism of chromosome-wide silencing by Xist is poorly understood. While it is established that the 5' region of Xist RNA, comprising the A-repeats and holding 7.5-8.5 copies of a conserved 26-mer sequence, is essential for Xist-mediated silencing, high-resolution structural information for the A-repeats is not available. Here, we report the three-dimensional solution structure of a 14-mer hairpin in the 5' region of a human A-repeat. This hairpin is remarkably stable and adopts a novel AUCG tetraloop fold, the integrity of which is required for silencing. We show that, contrary to previous predictions, the 3' region of single or tandem A-repeats mediates duplex formation in vitro. Significantly, mutations in this region disrupt the inter-repeat duplex formation in vitro and abrogate the silencing function of Xist A-repeats in vivo. Our data suggest that the complete A-repeat region may be stabilized by inter-repeat duplex formation and, as such, may provide a platform for multimerization and specific recognition of the AUCG tetraloops by trans-acting factors.
X 染色体失活（XCI）在雌性哺乳动物中依赖于非编码 RNA X 失活特异性转录本（Xist）。Xist 介导的染色体广泛沉默的机制尚未完全了解。虽然已经确定 Xist RNA 的 5' 区域，包括 A 重复序列，并且含有 7.5-8.5 个保守的 26 个碱基序列的拷贝，对于 Xist 介导的沉默是必需的，但 A 重复序列的高分辨率结构信息尚不可用。在这里，我们报告了人类 A 重复序列 5' 区域中 14 个碱基发夹的三维溶液结构。这个发夹非常稳定，采用了一种新的 AUCG 四链环折叠，其完整性对于沉默是必需的。我们表明，与之前的预测相反，单链或串联 A 重复序列的 3' 区域在体外介导双链形成。重要的是，该区域的突变破坏了体外重复间双链的形成，并使 Xist A 重复序列在体内的沉默功能丧失。我们的数据表明，完整的 A 重复区可能通过重复间双链形成而稳定，并且可以作为多聚体化和反式作用因子对 AUCG 四链环特异性识别的平台。
