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.

Enhancer of rudimentary (ER) is a small protein that has a unique amino acid sequence and structure. Its highly conserved gene has been found in all eukaryotic kingdoms with the exception of fungi. ER was proposed to be involved in the metabolism of pyrimidines and was reported to act as a transcriptional repressor in a cell type‐specific manner. To further elucidate ER functions, we performed the yeast two‐hybrid screen of the human lung cDNA library for clones encoding proteins interacting with the human ER protein. The screen yielded polymerase δ interacting protein 46 or S6K1 Aly/REF‐like target (PDIP46/SKAR), a protein possessing one RNA recognition motif (RRM) and being a protein partner of both the p50 subunit of DNA polymerase δ and p70 ribosomal protein S6 kinase 1 (S6K1). This interaction was further confirmed in vitro by the glutathione S‐transferase‐ER pull‐down of a protein of 46 kDa from a nuclear extract from human cells which was identified as PDIP46/SKAR by tandem mass spectrometry. The bipartite region of PDIP46/SKAR interacting with ER comprising residues 274–421 encompasses the docking site for S6K1 within the RRM and two serines phosphorylated by S6K1. ER and both isoforms of PDIP46/SKAR share the same nuclear localization in the mammalian cells and their genes display a ubiquitous pattern of expression in a variety of human tissues, so the interaction between ER and PDIP46/SKAR has an opportunity to occur universally in mammalian cells. Because PDIP46/SKAR is involved in the regulation of cell growth its interaction with ER may suggest some function for ER in that control.

Enhancer of rudimentary homolog (Drosophila) (ERH) is a small, highly conserved, nuclear protein with a unique three‐dimensional structure, whose gene has been identified in animals, plants and protists, but not in fungi. Involvement of ERH in fundamental processes such as regulation of pyrimidine metabolism, cell cycle progression, transcription and cell growth control has been suggested. Here, employing a yeast two‐hybrid system, a glutathione S‐transferase pull‐down assay and tandem MS, we demonstrate that Ciz1 is a bona fide interactor of human ERH. Ciz1 is a nuclear zinc finger protein interacting with p21^Cip1/Waf1, a universal inhibitor of cyclin‐dependent kinases, and is a DNA replication factor. The region of Ciz1 necessary for the interaction with ERH spans residues 531–644, encompassing its first zinc finger motif. This region overlaps the p21^Cip1/Waf1‐binding site, suggesting that the interaction with ERH could block the binding of p21^Cip1/Waf1 by Ciz1 in the cell. When ERH and Ciz1 are coexpressed in HeLa cells, Ciz1 recruits ERH to DNA replication foci.

Low cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study. Uptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay. TAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID. TAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.

ERH is a small, highly evolutionarily conserved nuclear protein of unknown function. Its three-dimensional structure is absolutely unique and it can form a homodimer through a β sheet surface. ERH has been shown to interact, among others, with PDIP46/SKAR and Ciz1. When coexpressed with the latter protein, ERH accumulates in replication foci in the nucleus of HeLa cells. Here, we report that when ERH is coexpressed with PDIP46/SKAR in HeLa cells, it is recruited to nuclear speckles, and identify amino acid residues critical for targeting ERH to both these subnuclear structures. ERH H3A Q9A shows a diminished recruitment to nuclear speckles but it is recruited to replication foci. ERH E37A T51A is very poorly recruited to replication foci while still accumulating in nuclear speckles. Consequently, ERH H3A Q9A E37A T51A is recruited neither to nuclear speckles nor to replication foci. The lack of interactions of these three ERH forms with PDIP46/SKAR and/or Ciz1 was further confirmed in vitro by GST pull-down assay. The residues whose substitutions interfere with the accumulation in nuclear speckles are situated on the β sheet surface of ERH, indicating that only the monomer of ERH can interact with PDIP46/SKAR. Substitutions affecting the recruitment to replication foci map to the other side of ERH, near a long loop between the α1 and α2 helices, thus both the monomer and the dimer of ERH could interact with Ciz1. The construction of the ERH mutants not recruited to nuclear speckles or replication foci will facilitate further studies on ERH actions in these subnuclear structures.

ERH is a 100 to about 110 aa nuclear protein with unique primary and three-dimensional structures that are very conserved from simple eukaryotes to humans, albeit some species have lost its gene, with most higher fungi being a noteworthy example. Initially, studies on Drosophila melanogaster implied its function in pyrimidine metabolism. Subsequently, research on Xenopus laevis suggested that it acts as a transcriptional repressor. Finally, studies in humans pointed to a role in pre-mRNA splicing and in mitosis but further research, also in Caenorhabditis elegans and Schizosaccharomyces pombe, demonstrated its much broader activity, namely involvement in the biogenesis of mRNA, and miRNA, piRNA and some other ncRNAs, and in repressive heterochromatin formation. ERH interacts with numerous, mostly taxon-specific proteins, like Mmi1 and Mei2 in S. pombe, PID-3/PICS-1, TOST-1 and PID-1 in C. elegans, and DGCR8, CIZ1, PDIP46/SKAR and SAFB1/2 in humans. There are, however, some common themes in this wide range of processes and partners, such as: (a) ERH homodimerizes to form a scaffold for several complexes involved in the metabolism of nucleic acids, (b) all these RNAs are RNA polymerase II transcripts, (c) pre-mRNAs, whose splicing depends on ERH, are enriched in transcripts of DNA damage response and DNA metabolism genes, and (d) heterochromatin is formed to silence unwanted transcription, e.g., from repetitive elements. Thus, it seems that ERH has been adopted for various pathways that serve to maintain genome integrity.