PROJECT SUMMARY/ABSTRACT Pontocerebellar Hypoplasia Type 1b (PCH1b) is an autosomal recessive neurological disorder characterized by hypoplasia/atrophy of the cerebellum and pons that is often fatal within the first year of life. The cerebellum and pons integrate information from sensory systems, the spinal cord, and other parts of the brain to regulate motor movements, breathing, and learning motor behavior. Individuals with PCH1b show muscle atrophy/weakness, microcephaly, and developmental delay. Most individuals with PCH1b do not live past childhood and current treatment is purely palliative. Mutations that cause PCH1b occur in the EXOSC3 gene, which encodes a structural cap subunit of an evolutionarily conserved and ubiquitously expressed RNA processing complex, the RNA exosome. The RNA exosome is a ribonuclease composed of both structural and catalytic subunits that play a critical role in the post-transcriptional regulation of RNA. This complex is required for 3’ to 5’ processing and degradation of a vast number of RNAs in both the nucleus and cytoplasm. Post-transcriptional processing of RNA is a critical regulatory step in gene expression, as underscored by the number of neurological diseases caused by defects in RNA processing factors. The tissue-specific phenotypes caused by the RNA exosome complex are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. Thus, we aim to investigate the in vivo functional consequences of distinct disease-causing amino acid substitutions in EXOSC3 that are linked to a range of mild to severe phenotypes in PCH1b. We have generated an allelic series of EXOSC3 disease-linked missense mutations in the Drosophila orthologue Rrp40 via CRISPR/Cas9 editing technology. Our previous work in flies revealed an enhanced requirement for Rrp40 in neurons. Furthermore, our RNA-seq analysis of brain-enriched transcriptomes of Rrp40 mutants revealed increases in steady-state levels of functionally important neuronal transcripts, suggesting that disease-causing amino acid changes in the Drosophila RNA exosome subunit Rrp40 contribute to neuronal dysfunction. Our goal now is to characterize how disease-causing amino acid substitutions in Rrp40 alter the molecular and cellular landscape of the developing nervous system in Drosophila in vivo. We will test the hypothesis that the RNA exosome regulates RNAs that are critical for proper neurodevelopment and function, a distinct subset of which are regulated by the RNA exosome cap subunit Rrp40 (EXOSC3) through three complementary aims: 1) Assess the functional consequences of amino acid substitutions in the Drosophila RNA exosome subunit Rrp40 corresponding to those that cause PCH1b; 2) Interrogate how Rrp40 mutations affect expression and localization of key neuronal transcripts within the fly brain; and 3) Exploit a genetic screen to determine whether aberrant accumulation of spe...