Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin

Abstract

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.

Transcripts of vaccinia virus postreplicative genes do not contain a 5’ methylguanosine cap

“…VLE mRNAs also contain 5' poly(A) leaders of variable length that are not complementary to the VLE DNA (27). We provided evidence that 5' poly(A) leaders of VLE mRNAs are formed by a mechanism similar to the synthesis of 5' poly(A) leaders of the VACV postreplicative transcripts (26). We further demonstrated that 5' highly polyadenylated VLE mRNAs do not contain the m 7 G cap moiety at their termini.…”

“…The exact biological role of the 5' poly(A) leaders in postreplicative transcripts has not been known for long, but it has been shown recently that the 5' poly(A) leaders provide viral mRNAs a translational advantage over the host cell transcripts in infected cells (25). We recently showed that VACV RNA polymerase belongs to the monophyletic group with RNA polymerases encoded by yeast virus-like elements (VLEs), and the VLE promoters are related to those of poxviruses (26). VLE mRNAs also contain 5' poly(A) leaders of variable length that are not complementary to the VLE DNA (27).…”

“…We further demonstrated that 5' highly polyadenylated VLE mRNAs do not contain the m 7 G cap moiety at their termini. Similarities between VACV and VLEs in terms of RNA polymerases, promoters and 5' mRNA poly(A) leaders (26,27) prompted us to perform a detailed characterization of VACV mRNAs at the level of individual mRNA molecules.…”

“…However, the presence of the 5' cap structure in individual VACV transcripts has never been tested. To achieve this goal, we decided to apply a modification of the rapid amplification of 5' cDNA ends (5' RACE) method that we successfully applied for analysis of 5' polyadenylation and capping of VLE mRNAs (26,27). We purified total RNA from HeLa cells infected and mock-infected with VACV and investigated the 5' ends of selected mRNAs covering all three GTCs.…”

Temporal Transcriptome and Promoter Architecture of the African Swine Fever Virus

“…A MEME 36 analysis identified and located a significant 19-nt motif (Figure 5a) located approximately 10 bp upstream of the TSS for 36 (out of 36) early gene promoter sequences, which we have called the Early Promoter Motif (EPM). The ASFV EPM is related to the VACV early gene promoter motif ('Upstream Control Element' or UCE) 37,38 as well as the yeast Virus-Like Elements (VLEs) promoters 39 (Supplementary Figure 5.c). Importantly, the distance distribution between the EPM and the TSS is limited to 9-10 bp (Figure 5b Figure 5d).…”