Interaction of 29,39-cAMP with Rbp47b plays a role in Stress Granule Formation

Abstract

29,39-cAMP is an intriguing small molecule that is conserved among different kingdoms. 29,39-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 29,39-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 29,39- cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 29,39-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 29,39-cAMP. Furthermore, SG formation was promoted in 29,39-cAMP-treated Arabidopsis seedlings, and interactions between 29,39-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 29,39-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.