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

Our analyses of the pGKL mRNA ends and results showing the importance of the UCR region for 5' mRNA end formation explain the surprising results of Schickel et al., who used a glucose dehydrogenase (gdh) reporter gene integrated into yeast cytoplasmic linear plasmids to measure the strength of several pGKL promoters. They showed that K2UCR6 controlling the gdh reporter results in almost two times higher expression than when the reporter is controlled by K2UCR10 even though these two UCRs have exactly the same length and UCS sequence (84). We found that 20% of K2ORF6 transcripts possess a 5' cap, whereas only uncapped mRNAs were found among K2ORF10 transcripts (Fig. 3C , Table S1A ). If a 5' cap substantially increases the translation efficiency of its transcript, differences in cap occurrence frequency can account for differences in the translatability of pGKL plasmid mRNAs. A 5' . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 cap positively affects the translation of mRNAs carrying a 5' poly(A) leader in a wheat germ extract (WGE) in vitro (85). Because mRNA capped with a N 7 -unmethylated cap is inefficient in translation initiation (86), a combination of our results and those of Schickel et al. (84) provides more indirect evidence for the N 7 methylation of 5'-capped pGKL mRNAs.