Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam, Germany.
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, 41092 Seville, Spain.
Plant Physiol. 2018 May;177(1):411-421. doi: 10.1104/pp.18.00285. Epub 2018 Apr 4.
2',3'-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2',3'-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2',3'-cAMP in Arabidopsis () protein complexes isolated from native lysate, suggesting that 2',3'-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2',3'-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 2',3'-cAMP. Furthermore, SG formation was promoted in 2',3'-cAMP-treated Arabidopsis seedlings, and interactions between 2',3'-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 2',3'-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.
2',3'-cAMP 是一种有趣的小分子，在不同的生物界中都有保守存在。2',3'-cAMP 大概是在 RNA 降解过程中产生的，在应激条件下细胞内水平会升高。此前，我们观察到 2',3'-cAMP 存在于拟南芥（Arabidopsis）天然裂解物中分离出的蛋白复合物中，这表明 2',3'-cAMP 在植物中可能有潜在的蛋白伴侣。在这里，亲和纯化实验表明 2',3'-cAMP 与应激颗粒（SG）蛋白组结合。SG 是由蛋白质和 mRNA 组成的聚集体，使细胞能够选择性地储存 mRNA，以备在应激（如热应激）时使用，此时翻译起始受到抑制。通过大小排阻色谱和亲和纯化分析，我们鉴定出 SG 的关键组成部分 Rbp47b 是 2',3'-cAMP 的潜在相互作用伙伴。此外，在 2',3'-cAMP 处理的拟南芥幼苗中促进了 SG 的形成，并且在体外使用微尺度热泳法证实了 2',3'-cAMP 与 Rbp47b、RRM2 和 RRM3 的 RNA 结合结构域之间的相互作用。综上所述，这些结果（1）描述了 SG 形成的新型小分子调节，（2）为 2',3'-cAMP 的生物学作用提供了证据，（3）展示了一种用于鉴定蛋白-代谢物相互作用物的原始生化途径。
