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Identification of amino acid residues of ERH required for its recruitment to nuclear speckles and replication foci in HeLa cells.

Banko MI, Krzyzanowski MK, Turcza P, Maniecka Z, Kulis M, Kozlowski P - PLoS ONE (2013)

Bottom Line: 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 construction of the ERH mutants not recruited to nuclear speckles or replication foci will facilitate further studies on ERH actions in these subnuclear structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

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.

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Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy.EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). A. ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. 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. C. ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. D. ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.
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pone-0074885-g003: Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy.EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). A. ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. 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. C. ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. D. ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.

Mentions: The single-substituted mutant phenotypes could be grouped into three classes (Table 1). Substitution of the majority (18) of the examined residues failed to affect the recruitment of ERH to nuclear speckles or replication foci (data not shown). These included T18, S24 and S47, whether changed to alanine or to aspartic acid. The double-substituted forms T18A S24A (Figure 3A) and T18D S24D (data not shown) also did not show changes in their accumulation in nuclear speckles or replication foci. Substitution of any of four residues (H3, Q9, R17 and D66) resulted in a weaker recruitment of ERH to nuclear speckles, with the strongest effect observed for the H3A and Q9A forms (data not shown). Substitution of any of another four residues (E37, H39, K41 and T51) resulted in a weaker recruitment of ERH to replication foci, with the strongest effect observed for the E37A and T51A forms (data not shown).


Identification of amino acid residues of ERH required for its recruitment to nuclear speckles and replication foci in HeLa cells.

Banko MI, Krzyzanowski MK, Turcza P, Maniecka Z, Kulis M, Kozlowski P - PLoS ONE (2013)

Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy.EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). A. ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. 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. C. ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. D. ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3755989&req=5

pone-0074885-g003: Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy.EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). A. ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. 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. C. ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. D. ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.
Mentions: The single-substituted mutant phenotypes could be grouped into three classes (Table 1). Substitution of the majority (18) of the examined residues failed to affect the recruitment of ERH to nuclear speckles or replication foci (data not shown). These included T18, S24 and S47, whether changed to alanine or to aspartic acid. The double-substituted forms T18A S24A (Figure 3A) and T18D S24D (data not shown) also did not show changes in their accumulation in nuclear speckles or replication foci. Substitution of any of four residues (H3, Q9, R17 and D66) resulted in a weaker recruitment of ERH to nuclear speckles, with the strongest effect observed for the H3A and Q9A forms (data not shown). Substitution of any of another four residues (E37, H39, K41 and T51) resulted in a weaker recruitment of ERH to replication foci, with the strongest effect observed for the E37A and T51A forms (data not shown).

Bottom Line: 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 construction of the ERH mutants not recruited to nuclear speckles or replication foci will facilitate further studies on ERH actions in these subnuclear structures.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

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.

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