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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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GST pull-down assay with substituted forms of human ERH.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.
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pone-0074885-g004: GST pull-down assay with substituted forms of human ERH.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.

Mentions: We previously showed that fragment L7 of human PDIP46/SKAR comprising residues 259-421 and fragment B of human Ciz1 comprising residues 531-644, both GST-tagged, were able to pull down FLAG-tagged wild-type human ERH expressed in bacteria [10,14]. To verify that the indicated amino acid residues of ERH are indeed required for the interactions with PDIP46/SKAR or Ciz1, we performed GST pull-down assay with the both double-substituted ERH forms and the quadruple-substituted form, all FLAG-tagged (Figure 4). GST-tagged PDIP46/SKAR[L7] was unable to pull-down H3A Q9A but it was capable of binding to E37A T51A, albeit the interaction was not as strong as that with wild-type ERH. Conversely, GST-tagged Ciz1[B] bound H3A Q9A similarly to wild-type ERH and was unable to pull down E37A T51A. Accordingly, H3A Q9A E37A T51A interacted neither with PDIP46/SKAR[L7] nor with Ciz1[B].


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)

GST pull-down assay with substituted forms of human ERH.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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755989&req=5

pone-0074885-g004: GST pull-down assay with substituted forms of human ERH.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.
Mentions: We previously showed that fragment L7 of human PDIP46/SKAR comprising residues 259-421 and fragment B of human Ciz1 comprising residues 531-644, both GST-tagged, were able to pull down FLAG-tagged wild-type human ERH expressed in bacteria [10,14]. To verify that the indicated amino acid residues of ERH are indeed required for the interactions with PDIP46/SKAR or Ciz1, we performed GST pull-down assay with the both double-substituted ERH forms and the quadruple-substituted form, all FLAG-tagged (Figure 4). GST-tagged PDIP46/SKAR[L7] was unable to pull-down H3A Q9A but it was capable of binding to E37A T51A, albeit the interaction was not as strong as that with wild-type ERH. Conversely, GST-tagged Ciz1[B] bound H3A Q9A similarly to wild-type ERH and was unable to pull down E37A T51A. Accordingly, H3A Q9A E37A T51A interacted neither with PDIP46/SKAR[L7] nor with Ciz1[B].

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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