Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland.
Nucleic Acids Res. 2011 Nov;39(21):9390-404. doi: 10.1093/nar/gkr630. Epub 2011 Aug 16.
Guanine-quadruplexes (G4) consist of non-canonical four-stranded helical arrangements of guanine-rich nucleic acid sequences. The bulky and thermodynamically stable features of G4 structures have been shown in many respects to affect normal nucleic acid metabolism. In vivo conversion of G4 structures to single-stranded nucleic acid requires specialized proteins with G4 destabilizing/unwinding activity. RHAU is a human DEAH-box RNA helicase that exhibits G4-RNA binding and resolving activity. In this study, we employed RIP-chip analysis to identify en masse RNAs associated with RHAU in vivo. Approximately 100 RNAs were found to be associated with RHAU and bioinformatics analysis revealed that the majority contained potential G4-forming sequences. Among the most abundant RNAs selectively enriched with RHAU, we identified the human telomerase RNA template TERC as a true target of RHAU. Remarkably, binding of RHAU to TERC depended on the presence of a stable G4 structure in the 5'-region of TERC, both in vivo and in vitro. RHAU was further found to associate with the telomerase holoenzyme via the 5'-region of TERC. Collectively, these results provide the first evidence that intramolecular G4-RNAs serve as physiologically relevant targets for RHAU. Furthermore, our results suggest the existence of alternatively folded forms of TERC in the fully assembled telomerase holoenyzme.
鸟嘌呤四链体（G4）由富含鸟嘌呤的核酸序列组成的非经典四链螺旋排列。G4 结构的庞大和热力学稳定特性已在许多方面表明会影响正常的核酸代谢。体内将 G4 结构转化为单链核酸需要具有 G4 去稳定/解旋活性的专门蛋白质。RHAU 是一种人类 DEAH 盒 RNA 解旋酶，具有 G4-RNA 结合和解析活性。在这项研究中，我们采用 RIP-chip 分析来鉴定体内与 RHAU 相关的大量 RNA。大约有 100 种 RNA 被发现与 RHAU 相关，生物信息学分析表明，大多数 RNA 包含潜在的 G4 形成序列。在与 RHAU 选择性富集的最丰富的 RNA 中，我们鉴定出人端粒酶 RNA 模板 TERC 为 RHAU 的真正靶标。值得注意的是，RHAU 与 TERC 的结合依赖于 TERC 5'-区域中稳定 G4 结构的存在，无论是在体内还是体外。还发现 RHAU 通过 TERC 的 5'-区域与端粒酶全酶相关联。总之，这些结果首次提供了证据，证明内源性 G4-RNA 是 RHAU 的生理相关靶标。此外，我们的结果表明，在完全组装的端粒酶全酶中，TERC 存在替代折叠形式。
