PROJECT SUMMARY This essay aims to understand a fundamental property of neurons: their ability to self-recognize and self- avoid. Self-avoidance is an essential aspect of a neuron's function as it ensures that branches from the same cell minimize their overlap while maximizing their interactions with branches from other cells. In mammals, at the core of this process is the generation of sufficient Protocadherin (Pcdh) protein isoform diversity such that essentially every neuron in the brain is differentially barcoded at its surface and therefore appears different to other neurons. The generation of Pcdh protein isoform diversity requires complex mechanisms of Pcdh transcriptional and pre-mRNA splicing such that distinct Pcdh mRNA molecules - bearing a different 5' end (variable exon) but an identical 3' end (constant exons) - are expressed in individual cells. Understanding how different Pcdh mRNA molecules are produced represents a long-term mystery in the field of neuroscience. Answering this fundamental mystery is key in illuminating the process of neuronal self-avoidance and represents the first essential step toward dissecting how dysregulation of this Pcdh mediated self-avoidance can lead to severe neurological disorders, such as for instance autism spectrum disorder and schizophrenia. Despite their critical function in the brain, however, limited progress has been made in understanding how Pcdh mRNAs are transcribed and properly spliced as general models of gene expression regulation have failed to recapitulate this complex mechanism and as the tools required to study it directly in vivo have lagged behind. In this proposal, we aim to (i) test a paradigm-shifting hypothesis of Pcdh RNA transcription and splicing based on alternative trans-splicing of variable and constant exons encoded in tandem on the same DNA strand - a mechanism that we propose to be regulated by the 3D genome topology of the Pcdh locus - and (ii) design technological innovations that will allow precise manipulation of the Pcdh gene cluster in vivo to test our hypothesis directly in neurons. These studies have the potential to illuminate the complex mechanism of the generation of Pcdh isoform diversity and its role in neuronal self-avoidance and wiring of healthy and disease brains. The findings from these studies are also poised to open up a new class of regulatory mechanisms of RNA processing reactions, previously unobserved and uncharacterized in mammals but that we speculate are utilized by cells to overcome challenging problems of pre-mRNA splicing associated with complex gene architectures.

PROJECT NARRATIVE Crucial to the wiring of neural circuits is the expression of sufficient Protocadherin (Pcdh) protein isoform diversity such that essentially every neuron in the brain is uniquely barcoded at its surface and therefore capable to self-recognize and self-avoid. This proposal aims to develop novel genomic, genetic and biochemical approaches to test a new model of Pcdh RNA splicing mechanism. The findings from this work will provide general principles of gene expression regulation of Pcdh genes and more broadly in vivo.