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

(A) Growth curves and production of the pGKL1 killer toxin from K. lactis IFO1267 (blue), IFO1267 pbp1Δ (red), IFO1267 lsm1Δ (yellow) and IFO1267 pbp1Δ pab1Δ (green) strains. All strains were cultivated in YPD medium at 28°C for approximately 80 hours, and their OD 600 nm and toxin production were monitored. Production of the pGKL1 killer toxin into culture medium was assayed by a well diffusion test on YPD agar plates with a lawn of the S. cerevisiae S6/1 sensitive strain (Fig. S1) . Toxin levels in a culture medium in early stationary phase (~51 hours), when all the cultures reached comparable OD 600 , are depicted. Killer zones were measured using 100x diluted filter-sterilized culture medium, and the average width of the killer zone per cell was calculated for each strain, plotted with standard deviation and statistically analyzed (** p < 0.01, * p < 0.05) (B) 5' RACE analysis of pGKL mRNAs from the wild-type and PAB1 deletion strains. 5' RACE analysis of K1ORF2 and K1ORF3 mRNAs was performed using total RNA prepared from K. lactis IFO1267 and its double-deletion mutant K. lactis IFO1267 pbp1Δ pab1Δ. The individual columns represent the following quantities: 1/ percentage of all transcripts containing 5' non-template adenosine nucleotides; 2/ percentage of all transcripts containing a 5' cap structure; 3/ the average number (shown as the mean and median) of non-template adenosine nucleotides per mRNA molecule; 4/ the number of 5' non-template adenosine nucleotides found in the corresponding mRNAs. Data related to the wild-type strain K. lactis IFO1267 are replicated from Fig. 3C . The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/325316 doi: bioRxiv preprint Figure S1 -Production of the pGKL1 killer toxin in early stationary phase