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.
Highlights
Pichia acaciae and Kluyveromyces lactis each contain two cytoplasmic virus-like elements (VLEs, known as linear plasmids); i.e. pPac1-1 (12.6 kb), pPac1-2 (6.8 kb) and pGKL2 (13.5 kb), pGKL1 (8.9 kb) respectively [1,2]
The rather wide-spread and extremely A/T rich yeast virus like elements (VLEs, termed linear plasmids) which encode toxic anticodon nucleases (ACNases) ensure autoselection in the cytoplasm by preventing functional nuclear capture of the cognate immunity genes, but how? When expressed in the nucleus, the mRNA of the VLE immunity genes is split into fragments to which poly(A) tails are added
VLE encoded immunity factors cannot be functionally expressed in the nucleus For the three known VLE encoded ACNase toxin complexes PaT, zymocin and DrT, immunity functions were proposed to be encoded by PaORF4, KlORF3 and DrORF5, respectively [16,17,20]
Summary
Pichia acaciae and Kluyveromyces lactis each contain two cytoplasmic virus-like elements (VLEs, known as linear plasmids); i.e. pPac (12.6 kb), pPac (6.8 kb) and pGKL2 (13.5 kb), pGKL1 (8.9 kb) respectively [1,2]. The respective larger elements display substantial similarities to each other in terms of organization and gene content They can exist without the smaller ones as they encode all proteins required for nucleus-independent cytoplasmic replication and maintenance [3]. One subunit in either zymocin or PaT is highly conserved; it carries chitin binding and chitinase domains that recognize cell wall associated chitin of target cells as primary toxin receptor for subsequent import and/or activation [5,6,7] In both zymocin and PaT, a rather hydrophobic stretch or subunit appears to manage membrane transfer of the cytotoxic subunits, PaOrf (encoded by pPac ORF2) and γ-toxin (encoded by pGKL1 ORF4). Since PaOrf but not γ-toxin evades a possible repair of the tRNA halves by cellular tRNA ligases, it was speculated that the presence of two cleavage sites might allow the excision of a di-nucleotide, rendering the target tRNA non-repairable [12,13,14,15]
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