A coding region segment is necessary, but not sufficient for rapid decay of the HIS3 mRNA in yeast.
In Saccharomyces cerevisiae, the HIS3 (encoding imidazoleglycerolphosphate dehydratase) mRNA is unstable (t1/2 = 7 min), whereas the ACT1 (encoding actin) mRNA is more stable (t1/2 = 30 min). To define determinants responsible for rapid mRNA decay, hybrid genes comprised of various regions of these two mRNAs were constructed, transformed into yeast on centromere-containing vectors, and the half-lives of the resultant chimeric mRNAs were measured. To examine whether the 3'-untranslated region (3'-UTR) of HIS3 can confer instability to the ACT1 mRNA, DNA encoding the 3'-UTR of ACT1 was replaced with the corresponding region of HIS3. The hybrid mRNA containing the HIS3 3'-UTR decayed at a rate similar to the endogenous ACT1 mRNA. The mRNA containing the HIS3 5'-UTR and most of the HIS3 coding region fused to an ACT1 3'-fragment was unstable, indicating that HIS3 instability determinants are located within the HIS3 5'-UTR or coding sequence. Deleting 411 nucleotides (nt) from the coding region of either HIS3 or the 5'-HIS3-ACT1-3' chimeric gene resulted in a three- to fourfold stabilization of the respective mRNAs. However, insertion of this 411-nt fragment in-frame into the entire ACT1 gene had no destabilizing effect on the resultant hybrid mRNA. We conclude that the instability determinants of HIS3 mRNA are complex, involving a coding region segment and, possibly, the 5'-UTR.
Herrick
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D
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
Jacobson
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A
D000199:Actins / Q000235:genetics*; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D000199:Actins; D005656:Fungal Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C021434:imidazoleglycerolphosphate dehydratase