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

) or the cap methyltransferase ABD1 (MTase, (63)) genes. We constructed yeast plasmids pYX212::NLS-K2ORF3 and pYX213::NLS-K2ORF3, which had been designed to produce K2Orf3p fused with an SV40 nuclear localization signal under the control of either a strong constitutive triose phosphate isomerase (TPI) promoter or a tightly regulated GAL1 promoter, respectively. These plasmids were introduced into the S. cerevisiae strains carrying either ceg1 ts or abd1 ts temperature-sensitive alleles (62, 63) . We detected no growth in any of the resulting yeast strains at the restrictive temperature (data not shown). Multiple reasons can explain such a result. K2Orf3p may need to be artificially forced to interact with the Cterminal domain of RNA polymerase II or may require another co-factor for its proper function. The former approach was used to study an N-terminal fragment of the vaccinia virus capping enzyme D1 (AAs 1-545) in yeast, where it was produced as a protein fused with part of the mouse capping enzyme Mce1 (AAs 211-597). The resulting fusion protein rescued the growth of yeast strains lacking genes coding for a TPase (cet1Δ) and a GTase (ceg1Δ) (61).