PROJECT SUMMARY Gene expression regulation is critical for maintaining cellular homeostasis. Dysregulation of genes disrupts normal cell physiology and very frequently leads to diseases such as cancer, developmental disorders, and neurodegenerative diseases. Gene regulation, however, occurs at different levels, and many genes are regulated post-transcriptionally at the RNA level. Two major methods of post-transcriptional gene regulation are RNA processing and RNA degradation, which function to regulate the steady-state level of different RNAs in the cell (quantity control) and to ensure the destruction of aberrant mRNAs that, if translated, would be deleterious to the cell (quality control). These processes involve the actions of enzymes called ribonucleases, and mutations in several of these enzymes have been found to cause human disease. Yet, the vast spectrum of enzymes involved in these processes has not been completely uncovered, and many rare inherited diseases have unknown underlying causes. Additionally, even proteins with known ribonuclease functions remain poorly characterized. Therefore, this project aims to investigate the roles of five proteins in messenger RNA (mRNA) processing and degradation, with the ultimate objective of better understanding these essential gene expression- regulating processes. The first goal of this proposal will be the identification of endonuclease targets and specific cleavage sites, which will be accomplished using a streamlined bioinformatics approach. Additionally, this study will identify the sequence-specific and/or structural determinants of endonuclease target recognition and cleavage, determine the impact of sub-cellular localization on target selection, investigate the enzymes' contributions to cellular homeostasis, and determine the physiological consequences of endonuclease dysregulation. Gaining molecular insight into the endonuclease functions of the proteins described in this study will fill considerable gaps in our current understanding of RNA regulatory networks. These discoveries could additionally reveal important links between the critical RNA control mechanisms of processing and degradation, and the manifestation of human disease.