Unconventional mRNA processing and cap-independent translation in yeast linear plasmids

Abstract

Linear plasmids have been identified in various yeast species, notably the Kluyveromyces lactis pGKL1/2 plasmids, which are cytoplasmic and serve as models for this class. These plasmids carry terminal proteins, compact genomes encoding 15 genes - including a killer toxin, RNA and DNA polymerases, a helicase, and a putative capping enzyme - though most gene functions remain unverified. We analyzed pGKL mRNAs using 5' and 3' RACE and hDcp2 decapping assays. Despite encoding a predicted capping enzyme, only a subset of plasmid transcripts are 5'-capped, and none are 3'-polyadenylated. Surprisingly, most pGKL-derived mRNAs begin with short, non-templated poly(A) sequences and lack 5' caps. To test the role of the poly(A)-binding protein (Pab1) in this process, we constructed a K. lactis strain with deletions in PAB1 and PBP1. These deletions had no effect on plasmid stability, 5' UTRs, or toxin production. Furthermore, several pGKL transcripts fail to bind the cap-binding translation factor eIF4E in vitro, unlike host mRNAs. Killer toxin expression from native pGKL mRNAs persists in eIF4E-depleted strains, while expression from a Pol II-driven construct ceases under the same conditions, indicating capindependent translation. Notably, the pGKL-encoded RNA polymerase and capping enzyme resemble those of vaccinia virus, as do the plasmid mRNAs' 5'-terminal poly(A) tracts, analogous to intermediate/late vaccinia transcripts. These findings suggest a convergent strategy for cap-independent gene expression in yeast linear plasmids and poxviruses.