Analysis of mRNA decapping and localization factors in translational regulation of autophagy genes

다국어 초록 (Multilingual Abstract)

Macroautophagy (here after autophagy) is a well-conserved cellular process that facilitates the degradation and recycling of cytoplasmic components. A double-membrane vesicle known as autophagosome is formed and fused to the vacuole/lysosome during autophagy. The transcriptional regulation of autophagy-related (ATG) genes have been studied under nutrient deprivation condition. However, little is known about their translational regulation. In a recent report, translation of Atg1 and Atg13, two proteins essential for autophagy, are regulated by Dhh1, a DEAD-box RNA helicase. A positive role of Dhh1 in translational regulation has been reported in a subset of mRNAs including STE12 mRNA. Dhh1 functions as an mRNA decapping activator in the mRNA decay pathway. mRNA decapping enzymes (Dcp1/Dcp2), Xrn1 exoribonylease, and decapping activator (Edc3, Scd6, Pat1) interact to form an mRNA granule known as P-bodies. Atg1 forms a protein kinase complex and is known to be phosphorylated by Atg13 depending on nutrient conditions. Atg13 plays a role in activating Atg17-31-29 trimer, which is maintained for the progress of autophagy, and is known as a hub protein.
In this study, we will investigate whether mRNA decapping and localization factors affect the translational regulation of Atg1 and Atg13 genes. Deletion mutation of DHH1, EDC3 and LOC1 will be analyzed under autophagy-inducing conditions those genes regulate the Atg1, Atg13 protein at the translational level. In addition, the expression of Ste12, affecting pseudohyphal growth, will be observed in Atg1 and Atg13 overexpression mutant.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 6
• 2.1. Strains, Media and cell culture conditions 6
• 2.2. Construction of HA-tagged Atg13 gene 9
• 2.3. Plasmid construction 9
• 2.4. Western blot analysis 11
• 2.5. Spotting assay under the autophagic stress condition 11
• 2.6. Microscopic observation 12
• 2.7. Filamentation assay 12
• 3. Results and discussions 13
• 3.1. Viability of PUF4, PUF5, PUF6, LOC1, EDC3 and SCD6 deletion Cells in nitrogen starvation. 13
• 3.2. Deletion of the decapping activator EDC3 or localization factor LOC1 increases the expression of ATG13. 15
• 3.3. Deletion of the decapping activator EDC3 and SCD6 increases the expression of ATG13. 17
• 3.4. Ste12 protein levels are decreased by ATG13 overexpression in filamentous-inducing condition. 19
• 3.5. ATG1 and ATG13 overexpression decreased filamentous growth on SLAD media. 22
• 4. Discussion 24
• 5. References 26
• ABSTRACT 33
• 감사의 글 35