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

Recent advances in the understanding of mRNA quality control and the function of closely associated cellular mRNA decapping enzymes allowed us to use S. pombe Rai1 and human Dcp2 proteins to directly investigate predicted N 7 -methylation at the 5' cap guanosine of pGKL mRNAs. Expression plasmids for the production of His-tagged Dcp2 and Rai1 proteins were obtained from Prof. Mike Kiledjian (Rutgers University) and Prof. Liang Tong (Columbia University), respectively. Both proteins were produced in the E. coli BL21(DE3) strain and purified to homogeneity exactly as described by Xiang S. et al. in 2009 (79) . We purified total RNA from the killer K. lactis IFO1267 strain for cap-methylation analysis. The total RNA was DNase I-treated and subsequently incubated with either purified Rai1 decapping enzyme, which removes unmethylated caps only (80), or purified hDcp2 decapping enzyme, which removes both methylated and unmethylated caps from the 5' ends of mRNAs (81,82). To control for the proper function of both mRNA decapping enzymes, we used in vitro-transcribed RNA corresponding to the first 579 nt (including 32 nt of the 5' UTR) of the K. lactis ACT gene, which encodes actin, (EnsemblFungi Id: KLLA0_D05357g) that had been capped using the vaccinia virus capping enzyme in either the presence or absence of SAM and subsequently incubated with Rai1 or hDcp2 decapping enzymes. The workflow of the experiment is illustrated in Fig. 4A . Treatment of K. lactis total RNA with hDcp2 significantly decreased the number of capped pGKL mRNAs represented by the originally highly capped transcripts from K2ORF1 and K2ORF2 genes, whereas treatment with Rai1 had no such effect. Identical results were obtained for the mRNA coding for actin (Fig. 4B ).