Project summary/abstract (as submitted for R00): RNA processing is an essential cellular process that when dysregulated underlies the development of neurological diseases. Several mutations in nuclease containing complexes cause pontocerebellar hypoplasia (PCH), a severe neurological disorder that often leads to prenatal death. Most cases of PCH are linked to mutations in the tRNA Splicing Endonuclease (TSEN) Complex, a heterotetramer responsible for the cleavage of tRNA introns prior to tRNA maturation, and its accessory protein, CLP1, which is a critical negative regulator of tRNA splicing. Genetic deletion of any single TSEN protein in yeast, engineered to have tRNAs without introns, was shown to be lethal, suggesting that the TSEN complex likely has substrates beyond the tRNAs, which may underlie the development of PCH. Likewise, mutations in many other RNA processing factors are also linked to PCH. To determine how mutations in certain RNA processing proteins such as CLP1 and TSEN protein lead to PCH, there remains a critical need to understand how these complexes assemble, are regulated, and how they recognize and process RNAs. Characterizing healthy cellular roles of these proteins is essential to determining how their dysfunction causes PCH. We aim to address these critical questions through the following proposed Aims. In Aim 1, we will determine how the CLP1/TSEN complex are regulated at the molecular and cellular level, how PCH mutations disrupt their regulation, and determine if abhorrent tRNA splicing products contribute to cell death, which may underlie PCH development. Further, in Aim 2, we will identify how PCH mutations alter the function and regulation of another RNA processing factor linked to PCH by determining how it specifically identifies and processes RNAs, how PCH mutations interfere with protein:protein interactions, and determining additional ways it may be regulated, using proteomics and molecular biology approaches. The proposed work is significant because it will provide mechanistic insight into how known PCH mutations may interfere with complex stability, function, or regulation for a range of PCH-linked proteins. This work will further provide insight into shared mechanisms by which these protein complexes cause PCH. Furthermore, the work here will characterize new RNA processing roles for these enzymes.