biostudies-literatureLama-Diaz TXunta de GaliciaXunta de Galicia/FEDER ‘Una manera de hacer Europa’Ministerio de Ciencia e InnovaciónCentro Singular de Investigación de GaliciaCIMUS receives financial support from the Xunta de Galicia/FEDERXunta de Galicia/FEDER 'Una manera de hacer Europa'6928-6944https://www.ebi.ac.uk/biostudies/studies/S-EPMC11229318biostudies-literatureUnknown52(12)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-deficNucleic acids researchAlternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase.PMC11229318PID2020-115472GB-I002019–2022ED481A-2018/0422019-2022ED431G 2019/02ED431C 2019/013Lama-Diaz TBlanco MGfalseAlternative 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 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-defic2024-01-01T00:00:00Z2024 Jul2025-04-04T11:25:20.557Z2025-04-04T11:25:20.557ZS-EPMC112293183878307410.1093/nar/gkae400