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Turnover rate of yeast PGK mRNA can be changed by specific alterations in its trailer structure
P Vreken1, R van der Veen, V C de Regt
1Biochemisch Laboratorium, Vrije Universiteit, Amsterdam, The Netherlands.
Abstract:
The effect of insertions in the 5'- and 3'-untranslated regions (UTR) of the Saccharomyces cerevisiae mRNA encoding phosphoglycerate kinase (PGK) on the stability of the transcript in vivo was determined. None of the structural alterations in the 5'-UTR affected mRNA turnover significantly, despite the strong negative effect on translational efficiency of some of these alterations previously observed. We conclude that the structure of the 5'-UTR is not important for the relatively high affinity of PGK mRNA in yeast cells. Moreover, translation cannot be a major factor in determining the rate of turnover of this mRNA. Insertion of either a polyG or polyU, but not a polyA or polyC, tract into the 3'-UTR of PGK mRNA increased its half-life by a factor of about two. Introduction of a hairpin structure containing 18 G.C base pairs had only a slight stabilizing effect. We argue that the stabilization by the structural changes in the 3'-UTR is due to altered folding of the mutant mRNA which retards a rate-limiting endonucleolytic cleavage step in the normal turnover pathway of PGK mRNA. The stabilizing effect of local structural alterations in the 3'-UTR opens the possibility for further increasing the product yield of a (heterologous) gene cloned in yeast cells.
Insights
Modifications to the 5' untranslated region (UTR) of yeast phosphoglycerate kinase (PGK) mRNA did not impact its stability. However, specific insertions in the 3' UTR significantly increased mRNA half-life, suggesting a method for enhancing gene expression in yeast.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Gene Expression Regulation
Background:
- The stability of messenger RNA (mRNA) is a critical determinant of gene expression levels.
- Untranslated regions (UTRs) of mRNA play significant roles in post-transcriptional regulation, including transcript stability and translation efficiency.
- Understanding mRNA turnover mechanisms in yeast is crucial for optimizing recombinant protein production.
Purpose of the Study:
- To investigate the impact of structural alterations in the 5' and 3' untranslated regions (UTRs) of Saccharomyces cerevisiae phosphoglycerate kinase (PGK) mRNA on its in vivo stability.
- To determine the role of mRNA structure and translation in the turnover rate of PGK mRNA.
- To explore the potential of manipulating 3' UTR sequences for enhancing gene product yield in yeast.
Main Methods:
- Site-directed mutagenesis was used to introduce various insertions and structural modifications into the 5' and 3' UTRs of PGK mRNA.
- The stability of wild-type and mutant PGK transcripts was assessed in vivo in Saccharomyces cerevisiae by measuring mRNA half-life.
- Translational efficiency of modified mRNAs was previously evaluated (though not detailed in this abstract).
Main Results:
- Structural alterations within the 5' UTR of PGK mRNA did not significantly affect its turnover rate, despite previously observed negative impacts on translational efficiency.
- Insertions of polyG or polyU tracts into the 3' UTR approximately doubled the half-life of PGK mRNA, while polyA or polyC insertions had no significant effect.
- A hairpin structure insertion in the 3' UTR conferred only a minor stabilizing effect on the mRNA.
Conclusions:
- The structure of the 5' UTR is not a major determinant of the stability of PGK mRNA in yeast.
- Translational efficiency does not appear to be a primary factor controlling the turnover rate of PGK mRNA.
- Stabilization of PGK mRNA by specific 3' UTR modifications likely results from altered mRNA folding that impedes rate-limiting endonucleolytic cleavage, offering a strategy to increase yields of cloned genes in yeast.