Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 6, D-14195 Berlin, Germany.
Max Planck Institute of Biochemistry, Structural Cell Biology Department, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Nucleic Acids Res. 2018 Mar 16;46(5):2648-2659. doi: 10.1093/nar/gky040.
The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.
UPF1 是 RNA 解旋酶，是无义介导的 mRNA 降解 (NMD) 途径的关键组成部分。先前的 UPF1 X 射线晶体结构阐明了其催化活性和调节的分子机制。在这项研究中，我们检查了 UPF1 核心的特征，并确定了一个结构元素，该元素在 UPF1 的各种核苷酸和 RNA 结合状态中采用不同的构象。我们使用生化和单分子测定证明，该结构元素调节 UPF1 的催化活性，因此我们将其称为调节环。有趣的是，哺乳动物中有两种选择性剪接的 UPF1 异构体，它们仅在调节环的长度上有所不同。异构体 1（UPF11）中的环比异构体 2长 11 个残基。我们发现，UPF11 中的这种小插入导致其易位和 ATP 酶活性增加两倍。为了确定这种差异催化活性的机制基础，我们已经确定了 UPF11 螺旋酶核心在apo 状态下的 X 射线晶体结构。我们的结果表明，这是一种调节 RNA 解旋酶的新机制，其中选择性剪接导致蛋白质内的细微结构重排，这对于调节酶的运动和催化活性至关重要。
