for van Hoof, Ambro R01 GM130147 PROJECT SUMMARY Post-transcriptional processing of RNA is a critical regulatory step in gene expression. Many evolutionarily conserved RNA processing enzymes mediate these key post-transcriptional events. This proposal focuses on molecular mechanisms linked to PontoCerebellar Hypoplasia (PCH), which serves as a paradigm for a growing number of neurological diseases caused by mutations in genes encoding RNA processing factors. PCH is a group of autosomal recessive neurodegenerative diseases characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. Mutations that cause PCH type 1 (PCH1) occur in genes that encode structural subunits of the RNA exosome (human Rrp40, 43, and 45), which plays critical roles in both RNA processing and degradation. Mutations that cause PCH types 2, 4, and 5 (PCH2/4/5) lie in genes that encode tRNA splicing endonuclease subunits (TSEN2, 15, 34, and 54). TSEN has a well-characterized role in tRNA processing but also other yet undefined functions. The subunits of these RNase complexes are all evolutionarily conserved and essential for viability. PCH1 mutations cause single amino acid substitutions that primarily occur in conserved residues. The discovery that mutations in multiple components of these complexes cause PCH strongly suggests that RNA processing dysfunction underlies PCH pathology. However, limited studies have assessed the functional consequences of these amino acid substitutions. Furthermore, given the common disease etiology, mutations in either the RNA exosome or TSEN complex could impair common RNA targets or classes of RNA targets, but the RNAs affected have not been systematically defined. These links to common biology strongly support our working hypothesis that mutations that cause PCH Types 1/2/4/5 impair the processing of a common set of RNA targets. Our previous collaborative efforts provide proof of principle that studies in model organisms can provide insight into how specific disease-causing amino acid substitutions impair RNA exosome function. Here we draw on our established collaboration and extensive preliminary data to perform a series of mechanistic studies in four aims.
Aim 1 assesses the functional consequences of amino acid changes that occur in PCH using budding yeast;
Aim 2 employs biochemical analysis in mouse cerebellum and cultured neuronal cells to define RNA exosome cofactors that could contribute to the tissue-specific nature of PCH;
Aim 3 couples studies in budding yeast and cultured neuronal cells to identify common RNA targets of the TSEN and RNA exosome complexes; and, finally, Aim 4 employs tissue-specific RNAi in Drosophila to begin to assess the requirement for specific RNA exosome cofactors and TSEN subunits in neurons. The long-term goal of this work is to fully define the function of these evolutionarily conserved RNase complexes while providing insight into molecular mechanisms that could contribute to neurological dysfunction in PCH.
Submission for van Hoof, Ambro R01 GM130147 PUBLIC HEALTH RELEVANCE The neurological disease Pontocerebellar hypoplasia causes defects in the formation of critical regions of the brain, the pons and the cerebellum, causing the children who are born with this condition to have a drastically shortened lifespan. The mutations that cause this devastating disease are located in genes that encode machinery critical for gene expression, but how the small changes that occur in these genes cause disease is not known. Here we propose studies designed to understand the molecular defects caused by these mutations. Project Narrative
