Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Mol Cell. 2021 Apr 15;81(8):1830-1840.e8. doi: 10.1016/j.molcel.2021.01.029. Epub 2021 Feb 12.
Translation of problematic mRNA sequences induces ribosome stalling, triggering quality-control events, including ribosome rescue and nascent polypeptide degradation. To define the timing and regulation of these processes, we developed a SunTag-based reporter to monitor translation of a problematic sequence (poly[A]) in real time on single mRNAs. Although poly(A)-containing mRNAs undergo continuous translation over the timescale of minutes to hours, ribosome load is increased by ∼3-fold compared to a control, reflecting long queues of ribosomes extending far upstream of the stall. We monitor the resolution of these queues in real time and find that ribosome rescue is very slow compared to both elongation and termination. Modulation of pause strength, collision frequency, and the collision sensor ZNF598 reveals how the dynamics of ribosome collisions and their recognition facilitate selective targeting for quality control. Our results establish that slow clearance of stalled ribosomes allows cells to distinguish between transient and deleterious stalls.
将有问题的 mRNA 序列翻译成简体中文会诱导核糖体停滞，触发质量控制事件，包括核糖体救援和新生多肽降解。为了定义这些过程的时间和调节，我们开发了一种基于 SunTag 的报告器，以实时监测单个 mRNA 上有问题的序列（多 A）的翻译。尽管含有多 A 的 mRNA 在数分钟到数小时的时间尺度上经历连续翻译，但与对照相比，核糖体负载增加了约 3 倍，反映出核糖体的长队列延伸到stall 之前很远的上游。我们实时监测这些队列的解析，并发现与延伸和终止相比，核糖体救援非常缓慢。暂停强度、碰撞频率和碰撞传感器 ZNF598 的调节揭示了核糖体碰撞的动力学及其识别如何促进选择性靶向质量控制。我们的结果表明，缓慢清除stalled 核糖体可以使细胞区分短暂和有害的 stall。
