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

Replacement of the wild-type UCR sequence of the K2ORF2 gene by the K1ORF1 UCR sequence (K1UCR1, Fig. 3A ) led to a remarkable switch from nearly fully capped and almost not 5'-polyadenylated transcripts (91.7% capped transcripts, average of added non-template adenosine nucleotides per mRNA molecule ≈ 0) to largely uncapped and 5'-polyadenylated transcripts (7.7% capped transcripts, average of added non-template adenosine nucleotides per mRNA molecule ≈ 5). These values correspond very well to the level of 5' capping and nontemplate polyadenylation of K1ORF1 mRNAs expressed from the pGKL1 plasmid under control of natural K1UCR1 (Fig. 2E ). The same trend was also observed for the transcripts of the resistance marker gene G418, transcription of which was controlled by K1UCR2. The majority of these transcripts were uncapped, but all of them were 5' polyadenylated in exactly the same manner as natural K1ORF2 transcripts. The same promoter had been used to drive both G418 marker gene and K1ORF2 transcription. Additionally, the average number of nontemplate adenosine residues in the 5' leader was comparable in both genes (G418 resistance and K1ORF2) controlled by K1UCR2. All these findings strongly support the hypothesis that differences in 5' end formation of yeast linear plasmid mRNAs are controlled by UCR sequences and are independent of the coding sequence of a gene and its location within the pGKL plasmid system.