Mechanistic insights into the role of the CPEB4 neuronspecific microexon in neurodevelopmental disorders

Abstract

[eng] Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of RNA-binding proteins that regulate cytoplasmic changes in poly(A) tail lengh and therefore mRNA-specific stability and translation. Since their discovery in meiotic maturation, the role of CPEBs has expanded to somatic tissues, where they are involved in a wide range of physiological and pathological processes including neuronal functions. During cell cycle, CPEB4 is regulated by phase separation controlled through phosphorylations. CPEB4 hyperphosphorylation by Cdk1 and ERK2 leads to condensate dissolution, resulting in monomeric CPEB4 that is able to promote mRNA polyadenylation and translation activation. On the other hand, unphosphorylated CPEB4 assembles translationally inactive cytoplasmic condensates that repress its targets by sequestration. However, in neurons, the molecular mechanisms governing translation regulation by CPEB4 are poorly understood. Recent data unraveled that neuronal CPEB4 contains a neuron-specific 8 aminoacid-long microexon of unknown function. The mis-splicing of CPEB4 microexon is linked to the onset of neurodevelopmental disorders such as idiopathic autism spectrum disorder (ASD) or schizophrenia (SCZ) as the result of reduced polyadenylation and protein levels of ASD and SCZ-risk genes. Such gene expression alterations lead, in turn, to neuroanatomical, electrophysiological and behavioral phenotypes associated to ASD and SCZ. These works causally link CPEB4 microexon mis-splicing to neurodevelopmental disorders, underscoring the biological relevance of this 8-aminoacid microexon in the brain. However, how the skipping of the microexon affects CPEB4 function and why neurons, unlike non-neuronal cells, require this microexon are questions that remain unanswered. In this work we have performed a characterization of the dynamics, composition and regulation of the condensates formed by the two neuronal isoforms of CPEB4, namely nCPEB4 (including the microexon) and nCPEB4Δ4 (lacking the microexon). Our results suggest that both isoforms show distinct propensities for phase separation in neuron-like cells in resting conditions. Importantly, a combination of cellular and in vitro assays indicates that the microexon is required to ensure a reversible regulation of nCPEB4 phase separation following neuronal depolarization. Our work suggests that the microexon is required for the regulation of CPEB4 upon neuronal depolarization, which seems distinct from the cell cycle mechanisms. Moreover, to better characterize the condensates formed by nCPEB4 isoforms, we analyzed the composition of nCPEB4 isoforms interactome and showed they have a complex composition. Altogether, our results provide a comprehensive understanding of the dynamics, composition and potential regulation of nCPEB4 condensates in neuronal cells, and propose a mechanistical view of how the splicing of the ASD and SCZ-associated microexon could alter these features.