Cajal body assembly mechanisms in vitro and in the context of neurodegenerative disease

点击翻译按钮获取中文摘要

PROPOSAL SUMMARY/ABSTRACT Background and Central Hypothesis: Cajal bodies (CBs) are non-membranous nuclear organelles that are responsible for assembling small nuclear ribonucleoproteins (snRNPs) into functional complexes that can participate in pre-mRNA splicing. Two of the essential proteins of CBs – coilin and the survival of motor neuron protein (SMN) – are essential for vertebrate development and for CB integrity. A deficiency of SMN is the underlying cause of spinal muscular atrophy (SMA), a neurodegenerative disease caused by splicing defects, and SMN binds to coilin in the nucleus. Remarkably, the field lacks a mechanistic model for how these proteins participate in CB formation and function. Thus, we must ask whether a deficiency of SMN causes defects in CB formation and consequent failure of the final snRNP assembly steps. Because SMN is involved in early steps of snRNP assembly in the cytoplasm, it is difficult to separate this from its role in the nucleus. Therefore, I propose to study the mechanism of CB assembly by coilin and SMN in vitro. I will complement this with an in vivo study of CB composition and function in neurons and other tissues, using zebrafish as a powerful model system. Specific Aims: In aim 1, I will make use of biophysical techniques to measure the interactions between coilin, SMN, and CB RNA molecules. I have also developed a single molecule microscopy technique for studying the binding affinity and kinetics of these molecules using both bottom-up and top-down assembly. In aim 2, I will use zebrafish as a model organism for studying the effects of a genetic depletion of SMN on the CB interactome. Using wild-type and SMN -/- fish, I will compare two developmental time points using mass spectrometry to identify protein partners and enhanced UV-crosslinking immunoprecipitation and RNA-Seq (CLIP) for RNA partners. I will validate these results with confocal imaging.