Addressing the role of cold-induced RBM3 in stress granules and a connection to cytoprotection

• Applicant: Professor Dr. Florian Heyd
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 270986915

It has long been known that in mammals the RNA binding protein RBM3 is induced in conditions with reduced body temperature. This is clinically used in an approach called hypothermia, the controlled decrease of core body temperature. Hypothermia acts highly neuroprotective, and more generally cytoprotective, and is for example used as treatment in a condition called newborn hypoxic ischemic encephalopathy. It is also known that the beneficial effect of hypothermia is at least in part mediated through increased RBM3 expression. In previous work, we have uncovered temperature-controlled alternative splicing coupled to nonsense-mediated decay as the molecular mechanism that leads to increased RBM3 expression upon cooling. However, despite the widely-acknowledged cytoprotective function of RBM3 in diverse model systems and disease conditions, RBM3’s molecular mode of action remains incompletely understood. Based on extensive preliminary data, we suggest that a major part of RBM3’s cytoprotective function is mediated through its localization to stress granules (SGs), where it may act as RNA chaperone to protect RNAs from degradation and aggregation and may also influence liquid-to-solid phase transition. The overall goal of this proposal is to use in vitro, in cellulo and in vivo models to characterize the role of RBM3 in controlling physical properties of stress granules, the localization of RNAs to stress granules and an impact on their stability and translation, and how this connects to cytoprotection in various stress conditions. To do so, we will globally characterize RBM3-bound mRNAs by CLIP, follow polyadenylated RNAs using FISH, perform Ribo-Seq and pulse-chase metabolic labelling of RNAs followed by RNA-Seq, all as a function of RBM3 expression in different stress conditions. These global analyzes will be followed by validating selected individual examples. We will then map domains within RBM3 that are required for its function in SGs, also in vitro, and use a knockdown-rescue approach to connect RBM3’s function in SGs with its cytoprotective role. Cytoprotection and a connection to SGs will also be analyzed in disease-mimicking primary and ex vivo models and finally, we will establish a mouse line with constitutively increased RBM3 expression as a preclinical model to confirm our results in vivo. Altogether, this project will make a major contribution to the molecular-mechanistic understanding of the role of RBM3 in cytoprotection. In addition, the experiments will provide unprecedented insights and conceptual advances regarding mammalian RNA chaperones and their role in controlling SG biology and RNA stability, translation and aggregation in different stress conditions.

• DFG Programme: Research Grants