How dynamic interactions of RNA binding proteins control activity-dependent translation in neurons

Abstract The molecular processes of RNA transport and local translation are crucial in many aspects of neuronal biology, including axonal growth and synaptic plasticity. Although some RNAs – such as ACTB and ARC – have been well-studied and are known to be regulated in response to neural activation, the underlying mechanisms governing them in space and in time are poorly understood. RNA binding proteins (RBPs) are key regulators of RNAs, involved in all aspects of RNA biology from processing and nuclear export, to transport, translation, and degradation. Current estimations are that over 2,000 genes in the mammalian genome encode for RBPs, however, the molecular function of most of these predicted RBPs remains unknown. Understanding RBP function is challenging due to the complexity of RBP-RNA interactions. A single RBP can interact with thousands of target transcripts, and each transcript can be targeted by many RBPs. In addition, RBP-RNA interactions are dynamic – the association and dissociation of an RBP from its target can drive functional changes. This complex and dynamic nature suggest that RBPs do not function in isolation and are rather forming higher-order complexes to exert their functions. Here, I hypothesize that RBPs form functional ‘modules’, by interacting with each other directly or by binding on the same RNA molecule and propose a novel approach to study RBPs using a network perspective. I aim to characterize the network of RBP interactions and how it rewires following neuronal activity, in attempt to define functional modules and to identify the most promising candidate regulators. To do so, I propose to use a combination of approaches. One is an RNA- centric approach, focusing on two well-studied RNAs, discovering the complement of RBPs that bind them and comparing between them, followed by detailed follow-up experiments investigating whether and how those RBPs affect the target RNA (Aim 1 – K99). The other, a protein-centric global approach, studying RBP interactions in an unbiased manner and describing how they change in response to stimulation, in order to define regulatory modules and to identify candidate regulators for mechanistic follow up (Aim 2 – K99). The combined results of these studies will shed light on the architecture of RBP interactions and will define modules, which will advance our understanding of these crucial molecules and will set the stage to investigate their functions in depth in my independent lab (aim 3 - R00).