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

In Vitro Synthesis of Capped mRNA A DNA template corresponding to the first 579 nt (including 32 nt of 5' UTR) of the K. lactis ACT1 coding sequence (EnsemblFungi Id: KLLA0_D05357g) (Table S1B) was prepared by PCR with the forward primer modT7_KLactin_F (containing a modified T7 promoter sequence (Coleman et al., 2004) ) and the reverse primer actin_KL-rev2 using K. lactis IFO1267 total cDNA as a PCR template as follows: 5 min at 95°C; then 30 cycles of 30 sec at 94°C, 30 sec at 55°C, and 1 min at 72°C; and finally, 10 min at 72°C. PCR products were purified using a High Pure PCR Product Purification Kit (Roche) and in vitro-transcribed using a TranscriptAid T7 High Yield Transcription Kit (Fermentas). Template DNA was removed by a DNA-free TM Kit (Ambion). Transcribed RNA was purified by an RNA Clean & Concentrator-5 kit (Zymo Research), and 9 pmol of RNA was capped in vitro using the vaccinia virus mRNA capping enzyme (Ambion) in the presence or absence of SAM according to the manufacturer's instructions and purified using an RNA Clean & Concentrator-5 kit. Part of the sample was collected and analyzed by 5' RACE to determine the efficiency of the mRNA capping reaction, while the rest of the sample was used for decapping assays with Rai1 or hDcp2 mRNA decapping enzymes.