Szeto Jenn-Yeu A, Vega Mirella Vivoli, Mailliot Justine, Orriss George, Sun Lingling, Bufton Joshua C, Powers Kyle T, Yadav Sathish K N, Berger Imre, Schaffitzel Christiane
School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom.
School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom.
RNA. 2025 Jun 16;31(7):933-948. doi: 10.1261/rna.080300.124.
Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway involved in translational control and gene expression regulation. Core NMD factors up-frameshift proteins UPF1, UPF2, and UPF3B are conserved from yeast to humans and essential to target mRNAs with a premature stop codon for decay. UPF2 binding to UPF1 activates UPF1's ATPase and helicase activities, and UPF2 binding to UPF3B is important for its association with the exon junction complex and efficient NMD. However, UPF2's association with RNA remains largely uncharacterized. Here, we analyze nucleic acid binding, identifying the first and third MIF4G domains of UPF2 as main RNA-/DNA-binding modules. We find that UPF2's MIF4G domain-3 has RNA annealing activity, while full-length UPF2 unfolds our reporter hairpin RNA structure. We show that UPF2 preferentially binds and stabilizes single-stranded RNA (ss-RNA) in a sequence-independent manner. Concomitant to ss-RNA binding, UPF2 undergoes a distinct conformational change in its otherwise highly dynamic structure. UPF2's RNA binding and unfolding activity may support UPF1's helicase and messenger ribonucleoprotein remodeling activity and, in combination with UPF3B, stabilize UPF1's association with nonsense mRNA.
无义介导的mRNA降解（NMD）是一种参与翻译控制和基因表达调控的mRNA监测途径。核心NMD因子上移码蛋白UPF1、UPF2和UPF3B从酵母到人类都是保守的，对于靶向带有提前终止密码子的mRNA进行降解至关重要。UPF2与UPF1的结合激活了UPF1的ATP酶和螺旋酶活性，而UPF2与UPF3B的结合对于其与外显子连接复合体的关联以及有效的NMD至关重要。然而，UPF2与RNA的关联在很大程度上仍未得到充分研究。在此，我们分析了核酸结合情况，确定UPF2的第一个和第三个MIF4G结构域是主要的RNA/DNA结合模块。我们发现UPF2的MIF4G结构域3具有RNA退火活性，而全长UPF2会使我们的报告发卡RNA结构解折叠。我们表明，UPF2以序列无关的方式优先结合并稳定单链RNA（ss-RNA）。与ss-RNA结合同时，UPF2在其原本高度动态的结构中发生明显的构象变化。UPF2的RNA结合和解折叠活性可能支持UPF1的螺旋酶和信使核糖核蛋白重塑活性，并与UPF3B一起稳定UPF1与无义mRNA的关联。
