title: Identification of amino acid residues of ERH required for its recruitment to nuclear speckles and replication foci in HeLa cells.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland.
authors.forename: Monika I
authors.initials: MI
authors.lastname: Banko
authors.forename: Marek K
authors.initials: MK
authors.lastname: Krzyzanowski
authors.forename: Paulina
authors.initials: P
authors.lastname: Turcza
authors.forename: Zuzanna
authors.initials: Z
authors.lastname: Maniecka
authors.forename: Marta
authors.initials: M
authors.lastname: Kulis
authors.forename: Piotr
authors.initials: P
authors.lastname: Kozlowski
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D022003:Cell Nucleus Structures / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C400423:Ciz1 protein, human; C484495:ERH protein, human; D009687:Nuclear Proteins; C495327:POLDIP3 protein, human; D016601:RNA-Binding Proteins; D014157:Transcription Factors