ERH Genes from Fission Yeasts Figure 5. Complementation of erh1D mutation and cross-species experiments. (A) Serial dilutions of cells were spotted on EMM2+ADE alone or EMM2+ADE supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.005% SDS agar plates and incubated for 5 days. Strains used: auxotrophic (ade2 leu2) erh1+ FY7269 transformed with pREP1 (ZBM1023), FY7269 erh1D derivative ZBM1020 transformed with pREP1 (ZBM1024), ZBM1020 transformed with pREP1/SpErh1p (ZBM1025), ZBM1020 transformed pREP1/SjErh1p (ZBM1026) and ZBM1020 transformed with pREP1/ HsERH (ZBM1027). (B) Intracellular localization of SpErh1p in human HeLa cells by confocal microscopy. Upper series of images, cells transfected with plasmid coding for EGFP-tagged human ERH (pEGFP-N1/ER) alone or cotransfected with plasmid coding for mCherry-tagged human Ciz1 (pmCherry- N1/Ciz1); lower series of images, cells transfected with plasmid coding for EGFP-tagged SpErh1p (pEGFP-N1/SpErh1p) alone or cotransfected with pmCherry-N1/Ciz1. Direct fluorescence of EGFP or mCherry was observed in live cells. (C) Yeast two-hybrid analysis with SpErh1p used as bait. The host S. cerevisiae L40 cells coexpressing human ERH and Ciz1, human ERH and PDIP46/SKAR (both pairs as positive controls), SpErh1p and Ciz1, and SpErh1p and PDIP46/SKAR were lysed and the activity of the lacZ reporter gene (conversion of X-gal to a blue precipitate represented here as a strong gray color of lysed cells) was determined. doi:10.1371/journal.pone.0049059.g005 S. cryophilus [21]. The identities observed here between the fission yeast ERHs are in perfect agreement with these values and correctly reflect the phylogenetic relationships, with S. cryophilus and S. octosporus as the closest relatives, S. pombe as their nearest relative, and S. japonicus as the most divergent member of the genus [21,23]. The fission yeast ERH genes have five (Sperh1+, Soerh1+ and Scerh1+) or six (Sjerh1+) introns, which is significantly higher than in most of their other genes (Figure 1A). On average, there is approx. one intron per gene, with nearly half of them being intronless in all four species, and in S. pombe only approx. 3% of genes have five or more introns [7,17,21]. In contrast to the relatively low conservation of the amino acid sequence, the positions of introns are nearly perfectly preserved in the four ERH genes (Figure 1A). However, this phenomenon is not restricted to this gene only. Of all introns in the spliced 1:1:1:1 orthologs, 81% share positions in all four species [21]. On the other hand, the fission yeast spliceosome seems to exhibit relatively low fidelity since during the cloning of Sperh1+ and Scerh1+ we detected incorrectly spliced transcripts (Figure 1D). Regarding the length, the incorrectly spliced introns do not differ from other fission yeast introns, which are within the range of 29–819 bp. Indeed, they are very close to the average intron length, 78 bp [7,17]. Apparently, it is rather challenging for the spliceosomal machinery to remove as many as five introns from a pre-mRNA molecule of a gene with a very short ORF (in this case 312 bp). Notably, during the original annotation of the S. pombe genome the ERH gene was not identified but the authors predicted that some of the genes could remain undiscovered at that time if they have either a highly spliced structure with small exons or an ORF shorter than 100 aa [7]. As a first step toward characterization of the S. pombe erh1+ gene we established its expression pattern in wild-type cells in a set of experimental conditions commonly studied in fission yeast. It turned out that this gene is expressed at a similar level in all conditions tested, regardless of the ploidy, growth stage, nutrient availability, and exposition to hyperosmotic stress (Figure 2A). In PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49059