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 findings pointing to the essentiality of the K2Orf3p MTase active site for linear plasmid maintenance in yeast immediately raised the question of whether the pGKL transcripts are capped. We analyzed the 5' ends of the pGKL transcripts using a modified rapid amplification of 5' cDNA ends (5' RACE) approach. Briefly, we purified total RNA from the K. lactis IFO1267 strain, treated it with DNase I, synthesized cDNA using SuperScript III reverse transcriptase and random primers, G-tailed the cDNA using dGTP and terminal deoxynucleotidyl transferase (TdT) and amplified the 5' regions corresponding to transcripts from individual ORFs by PCR with an anchored oligo(dC) primer and an appropriate genespecific primer (see primer sequences in Table S2 ). Reverse transcriptases, SuperScript III including, can overcome the 5'-5' triphosphate bond between the 5' m 7 G cap and the first nucleotide of nascent mRNA transcripts and show whether the mRNA is capped or not (69) (70) (71) . PCR fragments were verified by digestion with restriction endonucleases and cloned into a pCR4-TOPO plasmid. From 20 to 79 independent clones were sequenced and analyzed for each ORF. We used K. lactis ACT transcript (EnsemblFungi Id: KLLA0_D05357g), which . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 codes for actin and is transcribed by the RNA polymerase II from a nuclear gene as a control 5' capped mRNA.