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Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
Abstract:
Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs. To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation. Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation. Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site. Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity. Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites. Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA. Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S. cerevisiae.
Insights
Researchers identified a Saccharomyces cerevisiae mutant defective in polyadenylation, revealing PAP1
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Processing
Background:
- Eukaryotic mRNA 3' end maturation involves cleavage and polyadenylation of precursor mRNAs.
- Understanding mRNA 3' end processing mechanisms is crucial for gene expression regulation.
Purpose of the Study:
- To investigate the mechanism and function of mRNA 3' end processing.
- To identify and characterize genes involved in polyadenylation in Saccharomyces cerevisiae.
Main Methods:
- Isolation and genetic analysis of a temperature-sensitive polyadenylation-defective mutant in Saccharomyces cerevisiae.
- Biochemical analysis of cellular extracts to pinpoint the defect in polyadenylation.
- Molecular cloning and characterization of the wild-type PAP1 gene.
- In vivo analysis of mRNA levels and structure following temperature shift.
Main Results:
- A single mutation caused both polyadenylation defects and temperature sensitivity for growth.
- The defect occurs after pre-mRNA cleavage but before polyadenylation.
- The mutated gene, PAP1, encodes a known poly(A) polymerase.
- Mutant cells produced poly(A)-deficient mRNAs at the nonpermissive temperature, yet translation persisted.
Conclusions:
- The PAP1 gene is essential for mRNA 3' end formation in Saccharomyces cerevisiae.
- The pap1-1 mutation affects a post-cleavage step in polyadenylation.
- The persistence of translation despite poly(A)-deficient mRNA suggests complex regulatory mechanisms.