| Post-transcriptional regulation of RNA has increasingly been recognized as a means to regulate translation, both temporally and spatially. Regulation of RNA localization and RNA stability contribute to changes in cellular protein levels and cells' ability to respond to their environment. For example, subcellular localization of mRNAs and targeted mRNA degradation affect protein levels by regulating an mRNA's translational availability, while post-transcriptional modifications of RNAs can affect the stability of protein synthesis machinery. The work detailed in this thesis focuses on two different post-transcriptional mechanisms that directly impact cellular responses.;Subcellular localization of mRNA has increasingly been recognized in polarized cells. In neurons, mRNA localization becomes extremely important because of the great distances separating the cell body where mRNAs are generated and the termini of their cytoplasmic processes needed for physiological functions that underlie movement, sensation, learning, and memory. However, the ultimate fate of such localized neuronal mRNAs has not been tested. For neuronal systems, RNA degradation has been demonstrated in dendrites but not in axons. This thesis focuses on the mechanisms of RNA decay in axonal processes. My results show that the proteins involved in mRNA degradation (Upf1, CCR4, DCP1a, XRN1) and mRNA sequestration (TIA-1) from translational machinery localize to sensory axons. Colocalization between these proteins and mRNAs suggest that axons have the ability to locally degrade mRNAs. Indeed, axonal beta-actin mRNA shows reduced survival after exposure to Semaphorin 3A or MAG while exposure to NGF increases its survival in axons. Together, these and other data outlined in this thesis indicate that localized mRNAs are subjected to ligand dependent stabilization or destabilization likely through traditional RNA decay pathways.;In contrast to mRNAs, tRNAs and rRNAs that are part of the translational machinery are long-lived, due in part to post-transcriptional modifications that confer stability to the these RNAs by modifying one or more of their nucleobases. The most common post-translational RNA modification which also confers stability to RNA is pseudouridine [1]. There are two possible mechanisms that a family of psi synthases uses to isomerize uridine to pseudouridine in various non-coding RNAs. Following previous research on two pseudouridine synthases, deuterium labeling studies and MS analysis were used to discern whether the product of the broken adduct between TruA and a fluorinated substrate analog is hydrated in the same manner as another psi synthase, RluA. This work demonstrates that a representative psi synthase from a third family of psi synthases identically handles 5-fluorouridinylated substrate analogs to form two isomeric products. |
