Abstract
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 cross-linking and analysis of cDNAs (χCRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein–RNA interactions in vivo on a minute time-scale. Here, using χCRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurements reveal rapid changes in protein–RNA interactions within 1 min 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. χCRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein–RNA interactions.
Highlights
RNA-binding proteins play a key role in shaping gene expression profiles during stress, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression
Survival under stress is contingent on the ability to rapidly reprogram gene expression and, while this ability has been largely attributed to the activity of transcription factors, it is becoming increasingly clear that RNA-binding proteins (RBPs) play a primary role in shaping gene expression response profiles by modulating RNA processing and decay[2]
The methodological advances underpinning the development of χCRAC enabled us to glimpse the highly dynamic reprogramming of RBP–RNA interactions in response to stress
Summary
RNA-binding proteins play a key role in shaping gene expression profiles during stress, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. Due to prolonged UV-irradiation, cells are exposed to major additional stresses, such as DNA damage, which can confound the results and insert a bias toward RNA transcripts that are specific for the irradiation conditions To tackle these problems, we have improved the original CRAC protocol and developed a UV-irradiation device that cross-links proteins to RNA in vivo in seconds. We have improved the original CRAC protocol and developed a UV-irradiation device that cross-links proteins to RNA in vivo in seconds These advancements enabled us to perform quantitative time-resolved in vivo measurements of direct protein–RNA interactions at 1-min time-point resolution.
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