Department of Biochemistry and Chemistry, Westminster College, New Wilmington, PA, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
RNA Biol. 2023 Jan;20(1):469-481. doi: 10.1080/15476286.2023.2171760.
Histone mRNA degradation is controlled by the unique 3' stem-loop of histone mRNA and the stem-loop binding protein (SLBP). As part of this process, the 3' stem-loop is trimmed by the histone-specific 3' exonuclease (3'hExo) and uridylated by the terminal uridylyl transferase 7 (TUT7), creating partially degraded intermediates with short uridylations. The role of these uridylations in degradation is not fully understood. Our work examines changes in the stability of the ternary complex created by trimming and uridylation of the stem-loop to better understand the role of this process in the histone mRNA life cycle. In this study, we used fluorescence polarization and electrophoretic mobility shift assays to demonstrate that both SLBP and 3'hExo can bind to uridylated and partially degraded stem-loop intermediates, although with lower affinity. We further characterized this complex by performing 1-µs molecular dynamics simulations using the AMBER force field and Nanoscale Molecular Dynamics (NAMD). These simulations show that while uridylation helps maintain the overall shape of the stem-loop, the combination of uridylation and dephosphorylation of the TPNK motif in SLBP disrupts key RNA-protein interactions. They also demonstrate that uridylation allows 3'hExo to maintain contact with the stem-loop after partial degradation and plays a role in disrupting key base pairs in partially degraded histone mRNA intermediates. Together, these experiments and simulations suggest that trimming by 3'hExo, uridylation, and SLBP dephosphorylation weakens both RNA-protein interactions and the stem-loop itself. Our results further elucidate the role of uridylation and SLBP dephosphorylation in the early stages of histone mRNA degradation.
组蛋白 mRNA 的降解受组蛋白 mRNA 独特的 3'茎环结构和茎环结合蛋白 (SLBP) 的控制。在这个过程中，3' 茎环结构被组蛋白特异性 3' 外切酶 (3'hExo) 修剪，并被末端尿苷酰转移酶 7 (TUT7) 尿苷酰化，产生带有短尿苷酰化的部分降解中间产物。这些尿苷酰化在降解中的作用尚不完全清楚。我们的工作检查了修剪和尿苷酰化茎环后三元复合物稳定性的变化，以更好地了解该过程在组蛋白 mRNA 生命周期中的作用。在这项研究中，我们使用荧光偏振和电泳迁移率变动分析来证明 SLBP 和 3'hExo 都可以结合尿苷酰化和部分降解的茎环中间产物，尽管亲和力较低。我们进一步通过使用 AMBER 力场和 Nanoscale Molecular Dynamics (NAMD) 进行 1-µs 分子动力学模拟来表征这个复合物。这些模拟表明，虽然尿苷酰化有助于维持茎环的整体形状，但 SLBP 中 TPNK 模体的尿苷酰化和去磷酸化的组合会破坏关键的 RNA-蛋白相互作用。它们还表明，尿苷酰化允许 3'hExo 在部分降解后保持与茎环的接触，并在部分降解的组蛋白 mRNA 中间产物中破坏关键碱基对方面发挥作用。这些实验和模拟表明，3'hExo 的修剪、尿苷酰化和 SLBP 的去磷酸化削弱了 RNA-蛋白相互作用和茎环本身。我们的结果进一步阐明了尿苷酰化和 SLBP 去磷酸化在组蛋白 mRNA 降解早期阶段的作用。
