Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase

In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demons...

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Bibliographic Details
Published in:Nucleic acids research 2024-07, Vol.52 (12), p.6928-6944
Main Authors: Lama-Diaz, Tomas, Blanco, Miguel G
Format: Article
Language:English
Subjects:
Alleles
Codon, Initiator - genetics
DNA Helicases - genetics
DNA Helicases - metabolism
Gene regulation, Chromatin and Epigenetics
Mitochondria - enzymology
Mitochondria - genetics
Mutation
Peptide Chain Initiation, Translational
Protein Isoforms - genetics
Protein Isoforms - metabolism
Ribosomes - genetics
Ribosomes - metabolism
RNA, Messenger - genetics
RNA, Messenger - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
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Summary:In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific ATI mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This has crucial implications for the rational design of genuine separation-of-function alleles and provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkae400