Title

The Role of Translation Initiation in Nonsense-Mediated mRNA Decay in the Yeast Saccharomyces Cerevisiae: a Dissertation

Date

7-16-1999

UMMS Affiliation

Graduate School of Biomedical Sciences, Department of Molecular Genetics & Microbiology

Document Type

Dissertation, Doctoral

Subjects

Saccharomyces cerevisiae; RNA, Messenger; Academic Dissertations

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

mRNA decay is an important cellular process that regulates gene expression and is tightly linked to the process of translation. Many studies have illustrated the link between mRNA turnover and translation, indicating that mRNA decay is a cytoplasmic event. In order to investigate further the link between translation and turnover, seven mutants in translation initiation factors were analyzed for their effect on mRNA decay, including: i) three mutant alleles of the PRT1 gene (prt1-1, prt1-26 and prt1-63), which encodes a subunit of elF3; ii) sui1-1, which encodes the smallest subunit of elF3; iii) sui2-1, which encodes elF2; iv) GCN2c, which encodes the elF2 kinase, and v) cdc33-42, a mutant in the cap binding protein elF4E. The results demonstrate that the prt1-1 mutation results in stabilization of nonsense containing mRNAs without affecting the half-lives of most other mRNAs, a phenotype similar to a upf1Δ strain.

To identify substrates for the nonsense-mediated mRNA decay pathway, mRNA differential display analysis was performed using RNA prepared from prt1-1, PRT1, upf1Δ and UPF1 strains. Although the abundance of the HHF2 mRNA is increased in the mutant strains the half-life is unaffected. However, the mRNA half-life of the transcriptional regulator SPT10 was increased in the mutant strains indicating the SPT10 transcript is a substrate for the nonsense-mediated mRNA decay pathway. Further characterization of the SPT10 transcript showed that it is a substrate for this pathway because the initiator AUG is present in a poor translation initiation context which results in aberrant translation initiation. Finally, several other mRNAs, predicted to be substrates for the pathway based on the leaky scanning model, were subsequently shown to decay through this pathway. These transcripts included the REV7, STE50, and UBP7 mRNAs. The results from these experiments lay the groundwork for addressing the potential regulatory role of the nonsense-mediated mRNA decay pathway.

Comments

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