Translation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output ~10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a ~3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between transient and deleterious stalls, and that the human quality control apparatus predominantly targets the nascent protein rather than the mRNA.

中文翻译：

---

核糖体失速相关翻译质量控制的动态调节

有问题的mRNA序列的翻译诱导核糖体失速。失速位点碰撞的核糖体被细胞质量控制机制所识别，导致核糖体与mRNA分离，随后新生多肽降解，在某些生物体中，mRNA降解。但是，这些过程的时间和规则尚不清楚。我们开发了一个基于SunTag的报告子，以实时监控包含难以翻译的poly（A）片段的单个mRNA的翻译。这位记者总结了人类细胞中的先前发现，即内部的poly（A）延伸蛋白输出减少了约10倍，而mRNA水平相对不受影响。长期的翻译成像表明，在没有可检测到的mRNA切割的情况下，含有poly（A）的mRNA可以被可靠地翻译。然而，核糖体密度的定量分析显示，与对照mRNA相比，含poly（A）的mRNA上核糖体的数量增加了约3倍，这与许多停滞的核糖体的排列一致。使用单分子harringtonine径流实验，我们通过细胞质量控制机制实时观察了这些队列的分辨率，发现与延长和终止相比，救援非常缓慢。我们提出，停滞的核糖体清除速度非常慢，为细胞区分瞬时停转和有害停转提供了基础，而人类质量控制仪器主要针对的是新生蛋白质而不是mRNA。与许多停滞的核糖体的队列一致。使用单分子harringtonine径流实验，我们通过细胞质量控制机制实时观察了这些队列的分辨率，发现与延长和终止相比，救援非常缓慢。我们提出，停滞的核糖体清除速度非常慢，为细胞区分瞬时停转和有害停转提供了基础，而人类质量控制仪器主要针对的是新生蛋白质而不是mRNA。与许多停滞的核糖体的队列一致。使用单分子harringtonine径流实验，我们通过细胞质量控制机制实时观察了这些队列的分辨率，发现与延长和终止相比，救援非常缓慢。我们提出，停滞的核糖体的清除速度非常慢，为细胞区分瞬时停转和有害停转提供了基础，而人类质量控制仪器主要针对的是新生蛋白质而不是mRNA。