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

To investigate the possible effects of yeast PABP1 protein (Pab1) on the expression of pGKL genes, we deleted the PAB1 gene from K. lactis. Because no experimental data describing PAB1 deletion in the yeast K. lactis were available, we followed a strategy used for the deletion of PAB1 in the yeast S. cerevisiae (119). Deletion of PAB1 gene is lethal in S. cerevisiae (94,120) but this lethality can be suppressed by the simultaneous deletion of the gene coding for Pab1p-binding protein (Pbp1) (119). We disrupted the PBP1 gene in the chromosomal DNA of the K. lactis IFO1267 strain using a loxP-G418-loxP system (121) and obtained the K. lactis IFO1267 pbp1::G418 strain. The G418 cassette was removed from the genome by an artificially produced Cre-recombinase, providing the strain K. lactis IFO1267 pbp1Δ. We used the same strategy to introduce a PAB1 deletion and to obtain the required strain, K. lactis IFO1267 pbp1Δ pab1Δ. Another protein that stabilizes some viral 5' poly(A) tracts and thus may also affect translation of the respective mRNAs is Lsm1 (39). We disrupted the LSM1 gene in the K. lactis IFO1267 genome using a strategy similar to that described for PBP1 deletion. The K. lactis pbp1Δ pab1Δ double-mutant strain is viable but exhibits a slow-growth phenotype, similar to the reported behavior in S. cerevisiae (119).