title: Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.
abstract: Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Alene
authors.initials: A
authors.lastname: Kast
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Raphael
authors.initials: R
authors.lastname: Voges
authors.affiliation: Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schroth
authors.affiliation: Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.
authors.forename: Raffael
authors.initials: R
authors.lastname: Schaffrath
authors.affiliation: Fachgebiet Mikrobiologie, Universität Kassel, Kassel, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Klassen
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Meinhardt
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D055646:Killer Factors, Yeast / Q000235:genetics / Q000378:metabolism*; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism*
chemical_list: D055646:Killer Factors, Yeast; D012331:RNA, Fungal; D012260:Ribonucleases; C049345:anticodon nuclease