Expression analysis of RNA14, a gene involved in mRNA 3' end maturation in yeast: characterization of the rna14-5 mutant strain.

Abstract

In Saccharomyces cerevisiae, Rna14 protein is involved in both cleavage and polyadenylation of mRNA in the nucleus. Previous work has demonstrated that this protein is also localized in mitochondria. Moreover, all known rna14 mutants can be separated into two distinct classes: the poly(A)-negative class, which contains mutants that are deficient in mRNA 3'-end processing, and the poly(A)-positive class, which includes those mutants that are not impaired in any of the steps in mRNA metabolism investigated. This suggests that in addition to its involvement in mRNA polyadenylation, Rna14p could have a second function related to mitochondrial metabolism. Here we investigated the regulation of RNA14 by characterizing the rna14-5 mutant, which is the only poly(A)-positive allele that also overproduces the RNA14 mRNA. We showed that both deregulation of RNA14 transcription and modification of RNA14 mRNA stability contribute to the strong accumulation of the transcripts in this mutant. Surprisingly, the RNA14 promoter itself is not essential for this phenotype of the rna14-5 mutant. However, the 3' UTR of the mRNA is necessary for overproduction of the transcripts, although it is not sufficient to deregulate a reporter gene by itself. Site-directed mutagenesis experiments provided additional data suggesting that the rna14-5 mutation acts at the protein level rather than modifying the properties of the RNA14 transcripts themselves. A tentative model accounting for the data is discussed, in light of the proposed extranuclear function of the Rna14p and its mitochondrial localization.