The process of mRNA turnover is a critical component of the regulation of gene expression. In the past few years, a discrete set of pathways for the degradation of polyadenylated mRNAs, in eukaryotic cells have been described. The major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which primarily leads to a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic degradation. A critical step in the primary pathway is decapping, since it effectively terminates the mRNA's existence and is the site of numerous control inputs. I discuss the properties of the decapping enzyme and how its activity is regulated to give rise to differential mRNA turnover. The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. In this work, I demonstrate by several criteria that CCR4 and CAF1 encode critical components of the major cytoplasmic deadenylase in yeast. First, both Ccr4p and Caf1p are required for normal mRNA deadenylation in vivo. Second, both proteins localize to the cytoplasm. Third, Caf1p co-purifies with poly(A) specific exonuclease activity, and this activity is dependent on the presence of Ccr4p. Interestingly, because Ccr4p and Caf1p have been shown previously to interact with transcription factors, these results suggest an unexpected link between mRNA synthesis and turnover. Both the Ccr4 and Caf1 proteins have significant homology to known exonucleases, in this work I demonstrate by several criteria that CCR4 encodes the catalytic subunit of the deadenylase. First, over-expression of Ccr4p rescues the deadenylation defects of a caf1Δ strain, indicating that Caf1p is not essential for deadenylation. Second, purification of Ccr4p co-purifies with poly(A) specific exonuclease activity, and this activity is not dependent on the presence of Caf1p. Third, point mutants in predicted catalytic residues of the Ccr4p exonuclease domain result in deadenylation defects in vivo and in vitro. The strong conservation of Ccr4p and Caf1p in other eukaryotes suggests that they will function in the process of deadenylation in other organisms.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/289856 |
| Date | January 2001 |
| Creators | Tucker, Morgan Dean |
| Contributors | Parker, Roy |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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