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

TGA-114nts-AACTAAGTTACTGTGCTATATACA -114nts-= TCGTATTAGATATACCTAAAATGGATTATGGACAAATAGAAATTTATAACGATTATTTTAGAAATGTTGATAAGAAAACTATAAAATACACTCCCATGGAGTGCATAGAATATA pGKL2-ORF10 (3' RACE) pGKL2 (13218)TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGATTTTT (13277) #3 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #27 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #5 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #25 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #23 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #22 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #21 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #20 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #19 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #6 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #16 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #8 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #10 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #13 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGAT #1 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGA #9 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGA #14 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGA #15 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGA #2 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGA #4 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTGA #17 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTG #26 TAAATTATTATGTAACATTAAATATACATTTAACACTGTATGCTACTATAACTG The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/325316 doi: bioRxiv preprint (for pGKL2). Individual cDNA sequences corresponding to transcripts of a given ORF are aligned below the template DNA. After mRNA was isolated from K. lactis IFO1267, cDNA was prepared by SuperScript III Reverse Transcriptase (M-MuLV mutant; clone names begin with the number sign (#)) or by AMV Reverse Transcriptase (Finnzymes; clone names begin with the letter A; this enzyme was used only for K1ORF2, K1ORF3 and K2ORF5). In this comparison, we have also included clones obtained from the double-mutant strain K. lactis IFO1267 pbp1Δ pab1Δ (see text; clone names beginning with the letter P; this approach was used only for K1ORF2 and K1ORF3). The uniform distribution of the clones indicate that the reverse transcriptases and strains used for mRNA isolation/cDNA synthesis/clone preparation do not affect the results. The sequences of at least 20 independent clones were determined and aligned with the plasmid (template) DNA sequence of each ORF. Sequence sections that share sequence identity with the template plasmid DNA are labeled in red. Sequence sections that do not share sequence identity with the template plasmid DNA, thus containing non-template nucleotides of mRNA transcripts, are labeled in green. Guanosine residues that correspond to the original caps at the 5' ends of the mRNAs are underlined in black (G). All sequences are shown in the 5' to 3' transcription orientation regardless of their ORF's natural orientation in pGKL plasmids. (B) The 5' untranslated regions of the actin gene (KLLA0_D05357g) encoded by K. lactis IFO1267. (C) The 3' untranslated regions of K1ORF1, K2ORF5 and K2ORF10 encoded by pGKL plasmids. Template DNA with its stop codon (in the gray box) is shown for each ORF. Numbers in paren