+ Identification and Functional Analysis of the erh1 Gene Encoding Enhancer of Rudimentary Homolog from the Fission Yeast Schizosaccharomyces pombe Marek K. Krzyzanowski1, Ewa Kozlowska2, Piotr Kozlowski1* 1 Department of Molecular Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland, 2 Department of Immunology, Faculty of Biology, University of Warsaw, Warsaw, Poland Abstract The ERH gene encodes a highly conserved small nuclear protein with a unique amino acid sequence and three-dimensional structure but unknown function. The gene is present in animals, plants, and protists but to date has only been found in few fungi. Here we report that ERH homologs are also present in all four species from the genus Schizosaccharomyces, S. pombe, S. octosporus, S. cryophilus, and S. japonicus, which, however, are an exception in this respect among Ascomycota and Basidiomycota. The ERH protein sequence is moderately conserved within the genus (58% identity between S. pombe and S. japonicus), but the intron-rich genes have almost identical intron-exon organizations in all four species. In S. pombe, erh1+ is expressed at a roughly constant level during vegetative growth and adaptation to unfavorable conditions such as nutrient limitation and hyperosmotic stress caused by sorbitol. Erh1p localizes preferentially to the nucleus with the exception of the nucleolus, but is also present in the cytoplasm. Cells lacking erh1+ have an aberrant cell morphology and a comma-like shape when cultured to the stationary phase, and exhibit a delayed recovery from this phase followed by slower growth. Loss of erh1+ in an auxotrophic background results in enhanced arrest in the G1 phase following nutritional stress, and also leads to hypersensitivity to agents inducing hyperosmotic stress (sorbitol), inhibiting DNA replication (hydroxyurea), and destabilizing the plasma membrane (SDS); this hypersensitivity can be abolished by expression of S. pombe erh1+ and, to a lesser extent, S. japonicus erh1+ or human ERH. Erh1p fails to interact with the human Ciz1 and PDIP46/SKAR proteins, known molecular partners of human ERH. Our data suggest that in Schizosaccharomyces sp. erh1+ is non-essential for normal growth and Erh1p could play a role in response to adverse environmental conditions and in cell cycle regulation. Citation: Krzyzanowski MK, Kozlowska E, Kozlowski P (2012) Identification and Functional Analysis of the erh1+ Gene Encoding Enhancer of Rudimentary Homolog from the Fission Yeast Schizosaccharomyces pombe. PLoS ONE 7(11): e49059. doi:10.1371/journal.pone.0049059 Editor: Juan Mata, University of Cambridge, United Kingdom Received November 25, 2011; Accepted October 7, 2012; Published November 7, 2012 Copyright: 2012 Krzyzanowski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grant N N301 158535 from the Ministry of Science and Higher Education, Poland (to P.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pkozlowski@biol.uw.edu.pl Introduction The enhancer of rudimentary homolog (ERH) gene is present in genomes of most eukaryotic organisms including Animalia, Plantae and Protista with the noteworthy exception of Fungi in which it has not been described yet in such prominent model organisms as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Aspergillus nidulans [1–8]. A typical ERH gene codes for a small, approx. 100 amino acid long polypeptide. Its sequence is not similar to any other protein known but a comparison between species belonging to different higher taxa shows that it has been conserved during evolution, especially in Vertebrata in which the human and zebrafish Danio rerio ERH proteins differ only by one conservative substitution [3]. Analysis of ERH does not reveal any important sequence motifs including a subcellular localization signal, however, studying human ERH we found that it is present predominantly in the nucleus [9]. The exact function of ERH is unknown but some have been proposed. The gene was originally identified in Drosophila melanogaster in which its mutation augmented the truncated wings phenotype resulted from the hypomorphic mutation in another gene, rudimentary (r). The r gene codes for the first three enzymatic activities of the de novo pyrimidine nucleotide biosynthetic pathway and the effect was only seen in the r mutant background. Hence, the novel gene was named enhancer of rudimentary and its protein product was proposed to be involved in the regulation of the metabolism of pyrimidines [1,10]. Based on the in vitro phosphorylation of its protein by CK2 and the expression pattern of its mammalian homologs the same group also suggested that it could play a role in the cell cycle [3]. Later, following identification of the Xenopus laevis DCoH/PCD protein, a cell-type specific transcriptional cofactor as its molecular partner, ERH was indicated as a transcriptional repressor at least in some cells [4]. Also, we have shown that in all human cells ERH could interact with two proteins, a DNA replication factor Ciz1, and PDIP46/SKAR, a component of the exon junction complex involved in the regulation of cell growth, suggesting some functions of ERH in these fundamental processes [9,11–13]. The only well characterized feature of ERH so far is its high resolution three- dimensional structure. ERH is a single domain protein which in accordance with its unique amino acid sequence, exhibits a novel a+b protein fold with the overall topology b1-310-b2-a1-a2-b3-b4- a3 [5,14–16]. PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49059