Signal Transduction Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709.
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854.
Proc Natl Acad Sci U S A. 2020 Aug 11;117(32):19245-19253. doi: 10.1073/pnas.1922284117. Epub 2020 Jul 29.
Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., KO), which elevates cellular 5-InsP levels by two- to threefold (i.e., within the physiological rheostatic range). The KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.
调控酶介导的信使 RNA（mRNA）5'端去帽，通常使转录本降解，从而动态重塑转录组。例如，mRNA 5'端去帽的保护促进了加工（P）体的积累——无膜的生物分子凝聚物。这种 mRNA 的区室化将其暂时从可翻译池中移除；这些被抑制的转录本被稳定和储存，直到 P 体解体允许转录本重新进入细胞质。在这里，我们描述了肌醇焦磷酸信号分子 5-InsP（5-二磷酸肌醇 pentakisphosphate）对 mRNA 稳定性和 P 体动力学的调节。首先，我们证明了在体外，重组 NUDT3（Nudix [核苷二磷酸连接部分 X]-型水解酶 3）抑制 5-InsP 的脱帽作用。接下来，在完整的 HEK293 和 HCT116 细胞中，我们监测了一系列 NUDT3 mRNA 底物在 CRISPR-Cas9 敲除（二磷酸肌醇 pentakisphosphate 5-激酶 1 和 2，即 KO）后的稳定性，该 KO 细胞通过两到三倍（即在生理电阻范围内）提高细胞内 5-InsP 水平。KO 细胞表现出 NUDT3 mRNA 底物水平升高和 P 体丰度增加。在 KO 背景下，5-InsP 合成的药理学和遗传学衰减使 NUDT3 mRNA 底物水平和 P 体计数恢复到野生型细胞的水平。此外，将代谢稳定的 5-InsP 类似物包裹在脂质体中递送到野生型细胞中，提高了 NUDT3 mRNA 底物的水平，并增加了 P 体的丰度。鉴于细胞内 5-InsP 水平通常会根据生物能量环境的变化而波动，这种肌醇焦磷酸对 mRNA 结构的调控代表了一种转录后调控过程。这种对 P 体动力学的影响与干细胞分化、应激反应以及潜在的神经退行性疾病和衰老的改善有关。
