TRANS-3 · Beyond the chromosome: unravelling the interplay between inter-chromosomal genome architecture and mRNA biogenesis

In TRANS-3 we will develop the theoretical and experimental framework to understand inter-chromosomal genome structure and activity. Despite advances in sequencing chromosomes and mapping their three dimensional (3D) organization, a full picture of 3D genome structure that details how the borders of various chromosome territories functionally interface one another is still missing. As an analogy, our world map does not indicate most natural corridors or manmade infrastructure that connect countries to one another. Worse, we barely understand how such connections function. My recent work has identified one of the few functional inter-chromosomal (trans) DNA interactions known to date: a splicing factory involving over ten chromosomes and orchestrated by the muscle-specific protein RBM20 around its key target, the TTN pre-mRNA. I hypothesize that this exemplifies how mRNA biogenesis instructs the formation of trans-interacting chromatin domains (TIDs) around mRNA factories, nuclear compartments that facilitate gene regulation. We will test this general hypothesis by dissecting the mechanisms and function of a specific, disease-relevant model: the RBM20 mRNA splicing factory. We will then explore the global impact of these regulations. First (WP1) we will mechanistically assess how mRNA factories form and act through live imaging of the nuclear positioning and alternative splicing dynamics of an RBM20-regulated locus. Secondly (WP2) we will examine the physiological role of mRNA factories by studying the effects of disease-associated mutations in RBM20 and the TTN regulator GATA4, and of genetic variability in TTN regulatory regions. Finally (WP3) we will develop and deploy a novel pipeline to identify, validate, and study new TIDs and mRNA factories through the combination of molecular biology, bioinformatics, biochemistry, and single cell biology. In all, TRANS-3 will venture beyond the chromosome frontier towards a deeper understanding of nuclear structure-function.