Molecular mechanisms regulating tau pre-mRNA alternative splicing

In the nervous system alternative pre-mRNA splicing diversifies protein modular functions, thus playing an essential role in regulating synaptic function, and plasticity as well as development of neuronal circuitry. Misregulation of alternative splicing is associated with numerous neurological and neuromuscular disorders such as spinal muscular atrophy and frontotemporal dementia.;Microtubule associated protein Tau, is involved in stabilization of axonal microtubule assembly thus regulating several functions such as neurite elongation, establishment of neuronal polarity etc. The Tau protein binds to microtubules via microtubule binding repeat regions. One of the microtubule binding regions is encoded by alternatively spliced exon10. Inclusion of exon10 creates Tau protein isoforms with four microtubule binding regions (4R), while skipping leads to the formation of protein isoforms with three microtubule binding regions (3R). In a normal adult human brain the ratio of 4R:3R Tau protein isoforms is maintained at 1. Studies have identified mutations in the tau gene associated with neurodegenerative disease frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) which affects the splicing regulation of exon10. This alters the ratio 4R:3R tau protein isoforms in the patient brain. Thus changes in tau exon10 splicing can cause neurodegeneration.;My thesis aimed at studying the molecular mechanisms regulating tau exon10 splicing, by identifying novel splicing regulators of this event. To this end I have developed an expression cloning based strategy and screened human adult and fetal cDNA libraries for regulators of tau exon10 splicing. Using a RNA oligomer affinity pulldown coupled to tandem Mass spectroscopy I have identified proteins interacting and regulating an RNA secondary structure at the tau exon10 5' splice site, which has been shown to regulate exon10 splicing. Our function based expression cloning and biochemical RNA affinity pulldown approaches have identified several novel regulators of exon10 splicing. The identification of these novel splicing factors and cis-regulatory elements help further our understanding of sporadic forms of neurodegenerative diseases in which both Cis and Trans factors may function as potential disease modifiers. In this study the assays we have developed provides a framework to study other alternative splicing events, as well as identifying potential therapeutic targets to splicing events that are involved in disease development.