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Identification of Conserved MEL-28/ELYS Domains with Essential Roles in Nuclear Assembly and Chromosome Segregation.

Gómez-Saldivar G, Fernandez A, Hirano Y, Mauro M, Lai A, Ayuso C, Haraguchi T, Hiraoka Y, Piano F, Askjaer P - PLoS Genet. (2016)

Bottom Line: The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes.We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation.Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Developmental Biology (CABD), CSIC/Junta de Andalucia/Universidad Pablo de Olavide, Seville, Spain.

ABSTRACT
Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28's conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

No MeSH data available.


Related in: MedlinePlus

Mapping of ELYS localization domains.(A) Transiently transfected HeLa cells expressing full-length human ELYS (1–2275) or ELYS fragments fused to GFP (green in merge) were fixed and counterstained to visualize DNA (blue in merge). Maximum intensity projection of z-sections spanning the metaphase plate is shown for ELYS; other images represent single confocal sections. Scale bars, 10 μm. (B) Schematic representation of ELYS and analyzed fragments. Orange (aa. 1980–1989) boxes indicate AT-hook sequences. Truncations containing ELYS aa. residues 600–1101, 1851–2024 and/or residues 2034–2275 (blue shading) were efficiently imported.
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pgen.1006131.g007: Mapping of ELYS localization domains.(A) Transiently transfected HeLa cells expressing full-length human ELYS (1–2275) or ELYS fragments fused to GFP (green in merge) were fixed and counterstained to visualize DNA (blue in merge). Maximum intensity projection of z-sections spanning the metaphase plate is shown for ELYS; other images represent single confocal sections. Scale bars, 10 μm. (B) Schematic representation of ELYS and analyzed fragments. Orange (aa. 1980–1989) boxes indicate AT-hook sequences. Truncations containing ELYS aa. residues 600–1101, 1851–2024 and/or residues 2034–2275 (blue shading) were efficiently imported.

Mentions: The overall architecture of MEL-28/ELYS is similar throughout the metazoa (see schematic representations in Figs 2C and 7B). All metazoan MEL-28/ELYS homologs include an N-terminal β-propeller domain, a central α-helical domain, and a C-terminal domain that includes at least one AT hook. Crystal structure determination of the N-terminal domain of mammalian ELYS showed that it forms a seven bladed β-propeller structure with an extra loop decorating each of the propeller blades [15]. In human cells, the N-terminal 1018 amino acids of ELYS (which includes the β-propeller domain and the central α-helical domain but not the C-terminal AT hook) is sufficient to localize the protein to NPCs [15]. Mutational disruption of the conserved loop on blade 6 of the β-propeller domain (“loop2”) prevents the 1–1018 aa. fragment from localizing to the nuclear rim.


Identification of Conserved MEL-28/ELYS Domains with Essential Roles in Nuclear Assembly and Chromosome Segregation.

Gómez-Saldivar G, Fernandez A, Hirano Y, Mauro M, Lai A, Ayuso C, Haraguchi T, Hiraoka Y, Piano F, Askjaer P - PLoS Genet. (2016)

Mapping of ELYS localization domains.(A) Transiently transfected HeLa cells expressing full-length human ELYS (1–2275) or ELYS fragments fused to GFP (green in merge) were fixed and counterstained to visualize DNA (blue in merge). Maximum intensity projection of z-sections spanning the metaphase plate is shown for ELYS; other images represent single confocal sections. Scale bars, 10 μm. (B) Schematic representation of ELYS and analyzed fragments. Orange (aa. 1980–1989) boxes indicate AT-hook sequences. Truncations containing ELYS aa. residues 600–1101, 1851–2024 and/or residues 2034–2275 (blue shading) were efficiently imported.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920428&req=5

pgen.1006131.g007: Mapping of ELYS localization domains.(A) Transiently transfected HeLa cells expressing full-length human ELYS (1–2275) or ELYS fragments fused to GFP (green in merge) were fixed and counterstained to visualize DNA (blue in merge). Maximum intensity projection of z-sections spanning the metaphase plate is shown for ELYS; other images represent single confocal sections. Scale bars, 10 μm. (B) Schematic representation of ELYS and analyzed fragments. Orange (aa. 1980–1989) boxes indicate AT-hook sequences. Truncations containing ELYS aa. residues 600–1101, 1851–2024 and/or residues 2034–2275 (blue shading) were efficiently imported.
Mentions: The overall architecture of MEL-28/ELYS is similar throughout the metazoa (see schematic representations in Figs 2C and 7B). All metazoan MEL-28/ELYS homologs include an N-terminal β-propeller domain, a central α-helical domain, and a C-terminal domain that includes at least one AT hook. Crystal structure determination of the N-terminal domain of mammalian ELYS showed that it forms a seven bladed β-propeller structure with an extra loop decorating each of the propeller blades [15]. In human cells, the N-terminal 1018 amino acids of ELYS (which includes the β-propeller domain and the central α-helical domain but not the C-terminal AT hook) is sufficient to localize the protein to NPCs [15]. Mutational disruption of the conserved loop on blade 6 of the β-propeller domain (“loop2”) prevents the 1–1018 aa. fragment from localizing to the nuclear rim.

Bottom Line: The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes.We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation.Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Andalusian Center for Developmental Biology (CABD), CSIC/Junta de Andalucia/Universidad Pablo de Olavide, Seville, Spain.

ABSTRACT
Nucleoporins are the constituents of nuclear pore complexes (NPCs) and are essential regulators of nucleocytoplasmic transport, gene expression and genome stability. The nucleoporin MEL-28/ELYS plays a critical role in post-mitotic NPC reassembly through recruitment of the NUP107-160 subcomplex, and is required for correct segregation of mitotic chromosomes. Here we present a systematic functional and structural analysis of MEL-28 in C. elegans early development and human ELYS in cultured cells. We have identified functional domains responsible for nuclear envelope and kinetochore localization, chromatin binding, mitotic spindle matrix association and chromosome segregation. Surprisingly, we found that perturbations to MEL-28's conserved AT-hook domain do not affect MEL-28 localization although they disrupt MEL-28 function and delay cell cycle progression in a DNA damage checkpoint-dependent manner. Our analyses also uncover a novel meiotic role of MEL-28. Together, these results show that MEL-28 has conserved structural domains that are essential for its fundamental roles in NPC assembly and chromosome segregation.

No MeSH data available.


Related in: MedlinePlus