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Macroautophagy/autophagy is a highly conserved catabolic process by which cytoplasmic constituents are delivered to the vacuole/lysosome for degradation and recycling. Autophagy occurs at a basal level in all cells to prevent the accumulation of damaged proteins and organelles, thus playing a pivotal role in the quality control of cytoplasmic components and in the maintenance of cellular homeostasis. These processes also function as a survival mechanism employed by cells that can be rapidly upregulated under certain stress conditions, such as starvation, endoplasmic reticulum (ER) stress, and infections. The process of autophagy from initiation to closure is tightly executed and controlled by the concerted action of autophagy-related (Atg) proteins. To maintain cellular homeostasis and prevent pathologies, the induction and amplitude of autophagy activity are finely controlled through regulation of ATG gene expression. Although substantial progress has been made in characterizing transcriptional and post-translational regulation of ATG/Atg genes/proteins, little is known about the translational control of autophagy. In this dissertation, I report that Psp2, an RGG-motif-containing RNA binding protein, positively regulates autophagy through promoting the translation of Atg1 and Atg13, two proteins that are crucial in the initiation of autophagy. Under nitrogen-starvation conditions, Psp2 interacts with the 5' UTR of ATG1 and ATG13 transcripts in an RGG motif-dependent manner and with eIF4E and eIF4G2, components of the translation initiation machinery, to regulate the translation of these transcripts. Deletion of the PSP2 gene leads to a decrease in the synthesis of Atg1 and Atg13, which correlates with reduced autophagy activity and cell survival. Furthermore, deactivation of the methyltransferase Hmt1 constitutes a molecular switch that regulates Psp2 arginine methylation status as well as its mRNA binding activity in response to starvation. These results reveal a novel mechanism for how Atg proteins become upregulated to fulfill the increased demands of autophagy activity as part of translational reprogramming during stress conditions and help explain how ATG genes bypass the general block in protein translation that occurs during starvation. We also show for the first time that expression of Atg1 and Atg13 is regulated at the translational level. While studying post-transcriptional regulation of ATG transcripts, I discovered that the Ccr4-Not complex has bidirectional roles in regulating autophagy before and after nutrient deprivation. Under nutrient-rich conditions, Ccr4-Not directly binds to and deadenylates ATG1, ATG7 and ATG9 mRNA to promote their degradation, thus contributing to maintaining autophagy at the basal level. Deletion or conditional knockdown of CCR4 or POP2 led to an increase in these ATG mRNAs, and subsequent protein levels, which correlate with elevated autophagy activity. Upon nitrogen starvation, Ccr4-Not no longer associates with these ATG mRNAs and releases its repression. In contrast to its role as an autophagy repressor when nutrients are replete, Ccr4-Not positively regulates the expression of a slightly different subset of ATG genes encoding the core machinery of autophagy, and the complex is required for sufficient autophagy induction and activity. These results reveal that the Ccr4-Not complex is indispensable to maintain autophagy at the appropriate amplitude in both basal and stress conditions. |
