Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress

RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic CRAC, a UV cross-linking method that enabled us to quantitatively measure the dynamics of protein-RNA interactions in vivo on a minute time-scale. Here, using kinetic CRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurement reveal rapid changes in protein-RNA interactions within one minute following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. Kinetic CRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein-RNA interactions.RNA结合蛋白（RNA-binding proteins）在胁迫条件下塑造基因表达谱的过程中发挥关键作用，但目前对这些相互作用的动态特性及其如何影响基因表达动力学的了解仍十分有限。为解决这一研究问题，我们开发了动力学χCRAC（kinetic χCRAC）——一种紫外交联（UV cross-linking）方法，可实现分钟级时间尺度上体内蛋白质-RNA相互作用动力学的定量检测。本研究利用动力学χCRAC技术，检测了酵母转录终止因子Nab3在葡萄糖饥饿胁迫下的全局RNA结合动态变化。这些检测结果揭示，施加胁迫后1分钟内，蛋白质-RNA相互作用便发生了快速改变。在应激响应的早期阶段，Nab3的结合变化在很大程度上不依赖于转录速率的改变，这表明存在正交的转录调控机制。我们还发现了Nab3在抑制应激响应基因表达方面的功能。动力学χCRAC技术有望极大地增强我们对蛋白质-RNA相互作用体内动力学的认知。