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

(A) Design of the control experiment. Capping of synthetic actin RNA with an unmethylated 5' cap and 5' RACE analysis after Rai1 treatment are depicted. A PCR cassette consisting of a modified T7 promoter (130) and the first 579 nt (including 32 nt of 5' UTR) of the K. lactis ACT gene coding sequence was transcribed using T7 RNA polymerase. The resulting uncapped RNA molecules (1) were capped using a vaccinia virus capping enzyme in the absence (2) or presence of SAM (not shown). The RNAs were incubated with either Rai1 (3) or hDcp2 (not shown). The presence of either methylated (not shown) or unmethylated 5' caps . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 after each step was monitored by 5' RACE followed by the cloning and sequencing of individual cDNA clones. The graphical representation of the 5' RACE results is similar to those in Fig. 3A. (B) Tabular representation of the 5' RACE experiments using either total RNA prepared from K. lactis IFO1267 or synthetic K. lactis actin mRNA capped by a vaccinia virus mRNA capping enzyme in either the presence (m 7 G-actin) or absence (G-actin) of SAM. RNA preparations were analyzed untreated or treated with Rai1 or hDcp2. Numbers represent the fractions of capped RNAs from all the independent cDNA clones analyzed (numbers in brackets labeled with NAC ). Numbers labeled with an asterisk are replicated from Fig. 3C . NACnumber of analyzed clones. Electrophoretic analysis of semi-quantitative RT-PCR detecting control HGT1 mRNA (upper panel) and K2ORF5 mRNA (lower panel) in samples as follows: Lane 1 -glutathione Sepharose binding GST-eIF4E fusion protein in the presence of K. lactis IFO1267 total mRNA (input); Lane 2 -same as in line 1, but reaction performed without reverse transcriptase (negative control); Lane 3 -supernatant after the first washing step (unbound mRNA); Lanes 4, 5, and 6 -supernatant after the first, third and sixth washing steps, respectively (unbound mRNA); Lane 7 -glutathione Sepharose binding complex GST-eIF4E-mRNA after the sixth washing step (bound mRNA). M -GeneRuler 100 bp DNA Ladder Plus (Thermo Scientific). PCR was performed using Taq DNA polymerase and gene-specific primers listed in Table S2 .