ABSTRACT

RNA helicases contribute to diverse aspects of RNA metabolism through their functions in re-arranging RNA structures. Identification of the remodelling targets of RNA helicases is a critical step in elucidating their cellular functions. Here, we show that, in contrast to many other ribosome biogenesis factors, the DExD box ATPase DDX55 predominantly localizes to the nucleoplasm and we identify a nuclear localization signal within the C-terminal region of the protein. DDX55 associates with pre-ribosomal subunits in human cells and is required for maturation of large subunit pre-rRNAs. Interestingly, in vitro analyses show that DDX55 selectively associates with double-stranded RNA substrates, which also stimulate its ATPase activity, and our data suggest that the C-terminal region of DDX55 contributes to this substrate specificity. The C-terminal region of DDX55 is also necessary for recruitment of the helicase to pre-ribosomes and, using in vivo crosslinking, we reveal a binding site for DDX55 in helix H62 of the 28S ribosomal RNA. Taken together, these data highlight the importance of the C-terminal region of DDX55 in substrate specificity and recruitment, and identify domain IV as a potential remodelling target of DDX55 during LSU biogenesis.

摘要

RNA 解旋酶通过其重新排列 RNA 结构的功能对 RNA 代谢的各个方面做出贡献。鉴定 RNA 解旋酶的重塑目标是阐明其细胞功能的关键步骤。在这里，我们表明，与许多其他核糖体生物发生因子相比，DExD 盒 ATPase DDX55 主要定位于核质，我们在蛋白质的 C 端区域内确定了核定位信号。DDX55 与人类细胞中的前核糖体亚基相关联，是大亚基前 rRNA 成熟所必需的。有趣的是，体外分析表明 DDX55 选择性地与双链 RNA 底物结合，这也刺激其 ATPase 活性，我们的数据表明 DDX55 的 C 端区域有助于这种底物特异性。DDX55 的 C 端区域也是将解旋酶募集到前核糖体所必需的，并且使用体内交联，我们在 28S 核糖体 RNA 的螺旋 H62 中揭示了 DDX55 的结合位点。总之，这些数据突出了 DDX55 的 C 端区域在底物特异性和募集中的重要性，并将结构域 IV 确定为 LSU 生物发生过程中 DDX55 的潜在重塑目标。