Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada; Manitoba Institute for Materials, University of Manitoba, Winnipeg, Manitoba, Canada.
Biochim Biophys Acta Gen Subj. 2018 Sep;1862(9):1973-1979. doi: 10.1016/j.bbagen.2018.06.009. Epub 2018 Jun 12.
Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2'OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2'OH of loop nucleotides.
鸟嘌呤四链体可以在 DNA 和 RNA 中形成，并通过各种蛋白质相互作用影响许多生物过程。已经表明 DEAD 盒 RNA 解旋酶蛋白 DDX21 可以结合和重塑 RNA 四链体，但对于不同四链体物种的特异性知之甚少。先前的报告表明，DDX21 可能与端粒重复 RNA 四链体（TERRA）相互作用，TERRA 是端粒的一个组成部分，有助于端粒异染色质形成和端粒长度调节。在这里，我们报告说 DDX21 的 C 末端直接与 TERRA 相互作用。我们首次使用 2D 饱和转移差异 NMR 来绘制 RNA 物种上的蛋白质结合位点，并表明 DDX21 的四链体结合结构域主要与四链体的磷酸核糖骨架相互作用。此外，通过突变环核苷酸的 2'OH，我们可以大大降低 DDX21 对四链体的亲和力，表明四链体的识别和对 TERRA 的特异性是通过与环核苷酸的 2'OH 的相互作用介导的。
