<i>ERH</i> genes and ERH proteins from four <i>Schizosaccharomyces</i> species.

<p>(A) Intron-exon organization of <i>ERH</i> genes from <i>S. pombe</i> (<i>Sperh1⁺</i>), <i>S. octosporus</i> (<i>Soerh1⁺</i>), <i>S. cryophilus</i> (<i>Scerh1⁺</i>) and <i>S. japonicus</i> (<i>Sjerh1⁺</i>). Number in parentheses gives total length of CDS plus introns. Black blocks represent exons and are drawn to scale; numbers on top give their lengths in bp. Incisions with numbers indicate intron positions and their length in bp. Consecutive introns are labeled with Roman numerals. (B) Alignment of ERH amino acid sequences from <i>S. pombe</i> (SpErh1p), <i>S. octosporus</i> (SoErh1p), <i>S. cryophilus</i> (ScErh1p) and <i>S. japonicus</i> (SjErh1p). Human ERH is shown as a reference sequence. Numbering according to SpErh1p. Number in parentheses indicates the length of the protein. Dots indicate identical residues and blanks denote missing amino acids. Table shows percent identity of sequences. (C) Predicted three-dimensional structure of Sperh1p generated by SWISS-MODEL using coordinates for human ERH from Protein Data Bank (PDB identifier: 2nmlA). Protein images produced with UCSF Chimera. Helices α1 and α2 and loop α1-α2 in both proteins and the first (Q46) and last (D55) amino acid residues of loop α1-α2 in SpErh1p are indicated. (D) Intron IV-exon V junctions in <i>Sperh1⁺</i> and <i>Sjerh1⁺</i> and intron I-exon II junction in <i>Sperh1⁺</i>. Sequences of introns are italicized. The AG sequence of the 3′ splice site is underlined and the neighboring AG is denoted by lower case. For details see text.</p

Expression of <i>erh1⁺</i> and characterization of Erh1p in <i>S. pombe</i>.

<p>(A) Northern analysis of <i>erh1⁺</i> transcript. Total RNA from haploid strain FY12697 cultured to mid-log phase in YES, cultured to stationary phase in YES, subjected to nutritional stress in low-glucose EMM2, subjected to nutritional stress in EMM2-N or subjected to hyperosmotic stress in YES supplemented with 2 M sorbitol and a diploid constructed freshly by crossing strains FY12697 and FY7519, cultured to mid-log phase in YES or subjected to nutritional stress in EMM2-N, separated by 1.2% formaldehyde/agarose gel electrophoresis and stained with ethidium bromide (upper panel) followed by transfer to membrane and hybridization with radiolabeled <i>erh1⁺</i> cDNA (lower panel). (B) Intracellular localization of Erh1p. Upper series of images, cells expressing yEGFP-tagged Erh1p from pREP1 (strain ZBM1021) visualized with Nomarski Interference Contrast, nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence; lower series of images, cells expressing yEGFP-tagged Erh1p from <i>erh1⁺</i> chromosomal locus (strain ZBM1028), nuclei stained with DAPI and localization of yEGFP-tagged Erh1p determined by direct fluorescence. (C) Identification of two forms of Erh1p. Immunoprecipitates with anti-HA monoclonal antibody from lysed cells expressing 3HA-tagged Erh1p (strain ZBM1022) or negative control (strain ZBM1023) separated by 15% SDS/polyacrylamide gel electrophoresis and stained with silver. Protein molecular mass standard of 21.5 kDa is shown to the left. Both protein bands (p22 and p23) were identified by tandem mass spectrometry as Erh1p-3HA.</p

Complementation of <i>erh1Δ</i> mutation and cross-species experiments.

<p>(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 (ade⁻ leu⁻) <i>erh1⁺</i> FY7269 transformed with pREP1 (ZBM1023), FY7269 <i>erh1Δ</i> 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 <i>S. cerevisiae</i> 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 <i>lacZ</i> reporter gene (conversion of X-gal to a blue precipitate represented here as a strong gray color of lysed cells) was determined.</p

Effects of <i>erh1⁺</i> disruption in <i>S. pombe</i> on growth rate and cell morphology.

<p>(A) Growth curves for three <i>erh1⁺</i> strains (solid lines), prototrophic ZBM1004, auxotrophic (ade⁻ leu⁻) FY7269 and auxotrophic (ade⁻ leu⁻ ura⁻) FY7266 and three corresponding <i>erh1Δ</i> strains (dashed lines), ZBM1004 derivative ZBM1005, FY7269 derivative ZBM1020 and FY7266 derivative ZBM1030 in EMM2S minimal medium (left) or in YES rich medium (right). Cultures were inoculated to OD₆₀₀ of 0.1 from stationary cultures in the same medium. (B) Cell morphology by light microscopy with Hoffman Modulation Contrast. Images of strains as in (A) cultured to stationary phase in YES.</p

Effects of <i>erh1⁺</i> disruption on <i>S. pombe</i> sensitivity to stresses and on cell cycle progression.

<p>(A) Serial dilutions of cells were spotted on YES alone or YES supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.01% SDS agar plates and incubated for 5 days. Strains used: prototrophic <i>erh1⁺</i> ZBM1004 and its <i>erh1Δ</i> derivative ZBM1005, auxotrophic (ade⁻ leu⁻) <i>erh1⁺</i> FY7269 and its <i>erh1Δ</i> derivative ZBM1020, and auxotrophic (ade⁻ leu⁻ ura⁻) <i>erh1⁺</i> FY7266 and its <i>erh1Δ</i> derivative ZBM1030. (B) Cell cycle profiles following nutritional stress in nitrogen-free EMM2 minimal medium. Cells of strains as in (A) were stained with PI and sorted by flow cytometry. Upper series of profiles, cells before nutritional stress (in mid-log phase in YES); lower series of profiles, cells after 48-hour nutritional stress in EMM2-N. Peaks representing 1C and 2C DNA content are indicated. Numbers indicate the percentage of cells with a given DNA content.</p

GST pull-down assay with substituted forms of human ERH.

<p>Indicated FLAG-tagged ERH forms incubated with either GST-tagged fragment L7 of human PDIP46/SKAR (GST-PDIP46/SKAR[L7]) or GST-tagged fragment B of human Ciz1 (GST-Ciz1[B]) and detected by western blotting with anti-FLAG antibody followed by enhanced chemiluminescence reaction. PDIP46/SKAR does not interact with ERH H3A Q9A or ERH H3A Q9A E37A T51A, and Ciz1 does not interact with ERH E37A T51A or ERH H3A Q9A E37A T51A.</p

Amino acid residues of human ERH critical for its recruitment to nuclear speckles and replication foci.

<p>Three-dimensional structure of a monomer of ERH was produced by UCSF Chimera package using coordinates from Protein Data Bank (2nmlA) [30]. Four β strands (β1, β2, β3 and β4), three α helices (α1, α2 and α3) and the N- and C-termini are indicated. Critical residues are shown with their side chains in color and are labeled using a single-letter code and position number in the polypeptide chain. Residues involved in the recruitment to nuclear speckles (in red) and replication foci (in green) are situated on the β sheet and near the loop between helices α1 and α2, respectively.</p

Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy.

<p>EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). <b>A</b>. ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. <b>B</b>. ERH H3A Q9A is present not only in the nucleus but also in the cytoplasm, shows diminished recruitment to nuclear speckles but still accumulates in replication foci. <b>C</b>. ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. <b>D</b>. ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.</p