Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek

The artificial transfer of pGKL plasmids naturally occurring in K. lactis into S. cerevisiae is rare and difficult. In addition to other obstacles, the host mitochondria and pGKL plasmids in . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/325316 doi: bioRxiv preprint S. cerevisiae are incompatible, and the recipient strain thus must be devoid of mitochondrial DNA (ρ 0 ) (42). We prepared CWO4 ρ 0 strains solely bearing either wild-type CDC33 or its temperature-sensitive cdc33-1 and cdc33-42 alleles by their long-time cultivation in the presence of ethidium bromide. To facilitate the transfer of pGKL plasmids to the new strains and to preserve their isogenic genetic background, we performed cytoduction using an incomplete mating (105) of the CWO4 ρ 0 strains with a nuclear fusion-deficient (kar1) yeast strain (S. cerevisiae YAT547) carrying both pGKL1 and pGKL2 plasmids (42). We obtained isogenic ρ 0 and [pGKL1/2] S. cerevisiae strains on the CWO4 genetic background that differed only in their CDC33 alleles. All these strains contain both pGKL plasmids and display a killer phenotype (Fig. 6A ). We required a gene coding for a yeast single-subunit protein toxin to serve as a control for cap-dependent translation. We chose a thoroughly studied K1 killer toxin encoded by the M1 viral satellite dsRNA from the yeast S. cerevisiae.