ABSTRACT

Prp43 is a DEAH-box RNA helicase involved in both splicing and ribosome biogenesis. Its activities are directly stimulated by several co-activators that share a G-patch domain. The substrates of Prp43, its mechanism of action and the modes of interaction with and activation by G-patch proteins have been only partially characterized. We investigated how Pfa1 and PINX1, two G-patch proteins involved in ribosome biogenesis, interact with Prp43. We demonstrate that a protruding loop connecting the β4 and β5 strands of Prp43 OB fold is crucial for the binding of the G-patch domain of Pfa1. However, neither this loop nor the entire OB fold of Prp43 is essential for PINX1 binding. We conclude that the binding modes of Pfa1 and PINX1 G-patches to Prp43 are different. Nevertheless, stimulation of the ATPase and helicase activities of Prp43 by both full-length Pfa1 and PINX1 requires the β4-β5 loop. Moreover, we show that disruption of this loop completely abrogates Prp43 activity during yeast ribosome biogenesis but does not prevent its integration within pre-ribosomal particles. We propose that the β4-β5 loop plays a crucial role in the transmission of conformational changes induced by binding of the G-patch to Prp43 active site and substrate RNA.

摘要

Prp43 是一种 DEAH 盒 RNA 解旋酶，参与剪接和核糖体生物发生。它的活动直接受到几个共享 G-patch 域的共激活剂的刺激。Prp43 的底物、其作用机制以及与 G-patch 蛋白相互作用和激活的模式仅被部分表征。我们研究了 Pfa1 和 PINX1 这两种参与核糖体生物发生的 G 补丁蛋白如何与 Prp43 相互作用。我们证明连接 Prp43 OB 折叠的 β4 和 β5 链的突出环对于 Pfa1 的 G-patch 域的结合至关重要。然而，这个循环和 Prp43 的整个 OB 折叠都不是 PINX1 绑定所必需的。我们得出结论，Pfa1 和 PINX1 G-patches 与 Prp43 的结合模式是不同的。尽管如此，全长 Pfa1 和 PINX1 刺激 Prp43 的 ATP 酶和解旋酶活性需要 β4-β5 环。此外，我们表明，在酵母核糖体生物发生过程中，该循环的破坏完全取消了 Prp43 活性，但不会阻止其在前核糖体颗粒中的整合。我们提出 β4-β5 环在由 G 补丁与 Prp43 活性位点和底物 RNA 结合引起的构象变化的传递中起着至关重要的作用。