Background: Nuclear pore complexes (NPCs) are molecular gateways that orchestrate cargo between the cytoplasm and nucleus and regulate cellular processes such as, gene expression. We utilized the Ty1 retrotransposon of Saccharomyces cerevisiae (S. cerevisiae) to study retroviral integration because Ty1 and retroviruses, including human immunodeficiency virus (HIV-1), have similar replication cycles and structurally and functionally conserved integrase (IN) enzymes required for genomic insertion of retrotransposons and retroviruses. Ty1 IN interacts with subunits of RNA polymerase III to mediate insertion upstream of RNA Polymerase III transcribed genes such as tRNA genes. Since yeast tRNA genes are recruited to NPCs, we examined the role of NPC subunits, called nucleoporins (Nups), in Ty1 transposition.
Methods: We tested a panel of 19 Nup mutant yeast strains for Ty1 mobility defects by monitoring the formation of HIS+ colonies in Nup mutant strains carrying a plasmid with a HIS3 marked Ty1 element. To determine if the defects in Ty1 mobility in Nup mutants were due to differences in the availability of Ty1 transposition intermediates, we quantified Ty1 mRNA levels in these yeast strains by quantitative PCR (qPCR), Ty1 cDNA levels by southern blotting, and Ty1 Gag protein levels by western blotting. We also examined Ty1 nuclear localization by fluorescence microscopy and tRNA gene expression by qPCR. We monitored endogenous Ty1 insertion events in our panel of Nup mutant strains by PCR to analyze Ty1 integration levels upstream of the glycine tRNA gene SUF16, which is a hot spot for Ty1 targeting. Because the SUF16 locus is located within the pericentromere of chromosome III, which may have different targeting requirements than other chromosome regions, we also measured Ty1 insertion upstream of the serine SUP61 tRNA gene which is located on the right arm of chromosome III.
Results: Of the 19 mutants tested here, 11 (nup120Δ, sec13-1, nup170Δ, nup53Δ, nup188Δ, nup192-15, nup60Δ, mlp1Δ, mlp2Δ, nup159-1, pom34Δ) Nup mutant yeast strains had Ty1 mobility defects and altered Ty1 element insertion upstream of the SUF16 and SUP61 loci. Interestingly, but unexpectedly, we discovered that Nup mutant yeast strains that had wild type levels of Ty1 mobility (nup84Δ, nup133Δ, ndc1-4, nup42Δ, nup100Δ, pom152Δ, nup2Δ), also had impaired Ty1 element insertion upstream of SUF16 and SUP61. The nup60Δ strain had strikingly impaired levels of Ty1 mRNA resulting in minimal Ty1 Gag protein, and cDNA production whereas the remaining Nup mutant yeast strains had minimal defects in Ty1 transcription, translation, cDNA production or Ty1-IN localization. Taken together, our data suggests that the NPC may function as part of both the Ty1 element transcription and integration machinery. .