Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Abstract

Linear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids from Kluyveromyces lactis as a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5' and 3' RACE analysis of all the pGKL1,2 mRNAs and found them not 3' polyadenylated and containing mostly uncapped 5' poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5' capping and/or 5' polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence that K2ORF3 encodes an mRNA cap guanine-N 7 -methyltransferase and that 5' capped pGKL1,2 transcripts contain N 7 -methylated caps. Translation of pGKL1,2 transcripts is enhanced in lsm1Δ and pab1Δ strains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5' mRNA capping, degree of 5' non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses. doi: bioRxiv preprint 4 synthesized in a template-independent manner. We show that plasmid promoters directly determine the formation of the mRNA 5' end leaders and their capping, pointing to a possible role of the plasmid RNA polymerase in this unusual phenomenon. We also provide the first evidence that MTase is an essential part of the K2Orf3p enzyme and that capped pGKL mRNAs are methylated at the N 7 position of the terminal guanosine moiety. Using in vitro and in vivo approaches, we demonstrate that translation of plasmid-specific mRNAs is independent of major cellular cap-binding proteins, translation initiation factor 4E (eIF4E), and the Pab1 and Lsm1 proteins that were speculated to be possible translational enhancers and/or stabilizers of cellular and viral mRNAs bearing 5' poly(A) stretches (38,39). Our results thus suggest the existence of a novel translation initiation mechanism. Materials and Methods General Strategy for the Modification of pGKL Plasmids Using Homologous Recombination In VivoK. lactis IFO1267 was transformed with a PCR-generated fragment consisting of 5' and 3' ends homologous to the part of the pGKL plasmid to be modified, a non-homologous part that introduces mutation/s into a plasmid-specific protein coding sequence, and a gene encoding a resistance marker (typically against G418 or hygromycin B) whose transcription is driven by the UCR of K1ORF1 or K1ORF2. This type of construct was prepared by fusion PCR; for the organization of such a construct, see Fig. 2A. After transformation, yeast cells were plated onto selective agar media, and their DNA content was analyzed for the presence of the modified pGKL plasmid using agarose electrophoresis. Both modified and wild-type t...

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

“…Next, we wished to characterize transcription initiation of the VLE genes. Our previous 5′ RACE-PCR experiments had revealed 5′ cap structures on the VLE-specific mRNAs, likely synthetized by VLE-encoded K2ORF3p mRNA capping enzyme, and also the presence of non-templated 5′ poly(A) leaders of heterogeneous lengths in mRNAs of 12 pGKL genes except for K2ORF2 , K2ORF3 and K2ORF8 [ 36 ]. Interestingly, heterogeneous 5′ poly(A) leaders are a known feature of poxviral intermediate and late transcripts [ 37 , 38 ].…”

“…Therefore, we concluded that slippage of VLE RNAP at the initiation site was the mechanism responsible for 5′ polyadenylation of the transcripts. Moreover, the identified INR sequence constituted an independent DNA element, not influenced by the sequence of the gene because the pattern of the sequenced 5′ RACE-PCR clones for K1ORF2 transcripts was the same as for G418 R transcripts produced from the K1UCR2 with the wt INR [ 36 ].…”

“…Our previous 3′ RACE-PCR experiments had revealed the absence of 3′ poly(A) tails in mRNAs of all 15 pGKL ORFs [ 36 ]. To shed light on the mode of transcription termination of the VLEs, we tried to identify sequence/secondary structure elements/signals near the 3′ termini.…”

“…Our previous 5′ RACE-PCR experiments revealed short poly(A) leaders at the 5′ mRNA ends of most pGKL-encoded genes [ 36 ]. These 5′ poly(A) leaders were heterogeneous in length among individual transcripts (1–21 adenosines per molecule) and not complementary to the template DNA.…”

A versatile bulk electrotransfection protocol for mouse embryonic fibroblast and iPS cells

“…Cytosolic presence and activity of RNA polymerase III for transcription of exogenous DNA has been detected (20, 21). Also, it has been recently evidenced that mRNAs can be transcribed from plasmids in yeast cytoplasm (22). Although cytosolic expression of mRNA from exogenous DNA has not been explored, the current study provided evidences which indicate cytosolic presence of the transgene may be enough for the transgene expression.…”