Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
Nucleic Acids Res. 2012 May;40(9):4110-24. doi: 10.1093/nar/gkr1306. Epub 2012 Jan 11.
Human telomerase RNA (hTR) contains several guanine tracts at its 5'-end that can form a G4-quadruplex structure. Previous evidence suggests that a G4-quadruplex within this region disrupts the formation of an important structure within hTR known as the P1 helix, a critical element in defining the template boundary for reverse transcription. RNA associated with AU-rich element (RHAU) is an RNA helicase that has specificity for DNA and RNA G4-quadruplexes. Two recent studies identify a specific interaction between hTR and RHAU. Herein, we confirm this interaction and identify the minimally interacting RNA fragments. We demonstrate the existence of multiple quadruplex structures within the 5' region of hTR and find that these regions parallel the minimal sequences capable of RHAU interaction. We confirm the importance of the RHAU-specific motif in the interaction with hTR and demonstrate that the helicase activity of RHAU is sufficient to unwind the quadruplex and promote an interaction with 25 internal nucleotides to form a stable P1 helix. Furthermore, we have found that a 5'-terminal quadruplex persists following P1 helix formation that retains affinity for RHAU. Finally, we have investigated the functional implications of this interaction and demonstrated a reduction in average telomere length following RHAU knockdown by small interfering RNA (siRNA).
人端粒酶 RNA（hTR）在其 5'端含有几个鸟嘌呤链，可形成 G4-四链体结构。先前的证据表明，该区域内的 G4-四链体破坏了 hTR 中一种称为 P1 螺旋的重要结构的形成，P1 螺旋是确定逆转录模板边界的关键因素。富含 AU 元件的 RNA（RHAU）是一种 RNA 解旋酶，对 DNA 和 RNA G4-四链体具有特异性。最近的两项研究确定了 hTR 和 RHAU 之间的特异性相互作用。本文证实了这种相互作用，并确定了最小相互作用的 RNA 片段。我们证明了 hTR 5'区域内存在多个四链体结构，并且发现这些区域与能够与 RHAU 相互作用的最小序列平行。我们证实了 RHAU 特异性基序在与 hTR 相互作用中的重要性，并证明 RHAU 的解旋酶活性足以解旋四链体并促进与 25 个内部核苷酸的相互作用，形成稳定的 P1 螺旋。此外，我们发现 P1 螺旋形成后，5'端的四链体仍然存在，与 RHAU 保持亲和力。最后，我们研究了这种相互作用的功能意义，并通过小干扰 RNA（siRNA）证实了 RHAU 敲低后平均端粒长度的减少。
