We employed virus-like elements (VLEs) pGKL1,2 from Kluyveromyces lactis as a model to investigate the previously neglected transcriptome of the broader group of yeast cytoplasmic linear dsDNA VLEs. We performed 5' and 3' RACE analyses of all pGKL1,2 mRNAs and found them not 3' polyadenylated and containing frequently uncapped 5' poly(A) leaders that are not complementary to VLE genomic DNA. The degree of 5' capping and/or 5' mRNA polyadenylation is specific to each gene and is controlled by the corresponding promoter region. The expression of pGKL1,2 transcripts is independent of eIF4E and Pab1 and is enhanced in lsm1Δ and pab1Δ strains. We suggest a model of primitive pGKL1,2 gene expression regulation in which the degree of 5' mRNA capping and 5' non-template polyadenylation, together with the presence of negative regulators such as Pab1 and Lsm1, play important roles. Our data also support a hypothesis of a close relationship between yeast linear VLEs and poxviruses.

• Keywords
• Lsm1, Pab1, eIF4E, linear cytoplasmic plasmid, pGKL, poly(A) leader, poxvirus, virus-like element,
• Publication type
• Journal Article MeSH

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

• MeSH
• Cytoplasm MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Fungal Proteins genetics   MeSH
• Transcription, Genetic * MeSH
• Kluyveromyces genetics   MeSH
• Nucleic Acid Conformation MeSH
• Evolution, Molecular * MeSH
• Polyadenylation MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Fungal MeSH
• Response Elements * MeSH
• Base Sequence MeSH
• Sequence Homology MeSH
• RNA Stability MeSH
• Viruses genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Fungal Proteins MeSH

Virus-like particles (VLPs) were isolated from the yeast Wickerhamia fluorescens strain CCY61-1-1. The VLPs are approximately 42 nm in diameter and contain only one species of dsRNA molecule. The apparent length of the dsRNA determined by native agarose gel electrophoresis was 4.6 kbp. Analysis of protein content of the VLPs showed them to contain one major capsid protein with an apparent molar mass of 74.5 kDa.

• MeSH
• RNA, Double-Stranded isolation & purification   MeSH
• Electrophoresis, Agar Gel MeSH
• Capsid isolation & purification   MeSH
• Yeasts virology   MeSH
• Molecular Weight MeSH
• RNA, Viral isolation & purification   MeSH
• Totivirus genetics   isolation & purification   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Double-Stranded MeSH
• RNA, Viral MeSH