Affiliation:
1. Department of Biochemistry and Molecular Biology, CIMUS, Universidade de Santiago de Compostela-Instituto de Investigación Sanitaria (IDIS) , Santiago de Compostela, A Coruña 15782 , Spain
Abstract
Abstract
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.
Funder
Ministerio de Ciencia e Innovación
Xunta de Galicia/FEDER ‘Una manera de hacer Europa’
CIMUS receives financial support from the Xunta de Galicia/FEDER
Centro Singular de Investigación de Galicia
Xunta de Galicia
Publisher
Oxford University Press (OUP)
Cited by
1 articles.
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