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A yeast two-hybrid screen for SYP-3 interactors identifies SYP-4, a component required for synaptonemal complex assembly and chiasma formation in Caenorhabditis elegans meiosis.

Smolikov S, Schild-Prüfert K, Colaiácovo MP - PLoS Genet. (2009)

Bottom Line: SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes.The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality.Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The proper assembly of the synaptonemal complex (SC) between homologs is critical to ensure accurate meiotic chromosome segregation. The SC is a meiotic tripartite structure present from yeast to humans, comprised of proteins assembled along the axes of the chromosomes and central region (CR) proteins that bridge the two chromosome axes. Here we identify SYP-4 as a novel structural component of the SC in Caenorhabditis elegans. SYP-4 interacts in a yeast two-hybrid assay with SYP-3, one of components of the CR of the SC, and is localized at the interface between homologs during meiosis. SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes. In the absence of SYP-4, the levels of recombination intermediates, as indicated by RAD-51 foci, are elevated in mid-prophase nuclei, and crossover recombination events are significantly reduced. The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality. Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly.

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Extended transition zone chromosome morphology and lack of chiasmata in meiotic prophase I nuclei in syp-4 mutants.(A) Schematic representation of the predicted SYP-4 protein. The region deleted in the tm2713 mutant allele is indicated (codons 28 through 84). Coiled-coil domains, with start and end points identified through the COILS program are shaded in dark gray. The black bar indicates the N-terminal region used for antibody production. (B) Low magnification images of DAPI-stained nuclei of whole-mount gonads from age-matched wild type and syp-4(tm2713) adult hermaphrodites. The extended transition zone chromosome configuration is observed until late pachytene in the latter. Progression from early to mid-prophase is observed from left to right. (C–H) High magnification images of DAPI-stained nuclei at transition zone (C,F), pachytene (D,G), and diakinesis (E,G) in wild type (C–E) and syp-4(tm2713) mutants (F–H). Bars, 5 µm (B) and 2 µm (C–H).
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pgen-1000669-g002: Extended transition zone chromosome morphology and lack of chiasmata in meiotic prophase I nuclei in syp-4 mutants.(A) Schematic representation of the predicted SYP-4 protein. The region deleted in the tm2713 mutant allele is indicated (codons 28 through 84). Coiled-coil domains, with start and end points identified through the COILS program are shaded in dark gray. The black bar indicates the N-terminal region used for antibody production. (B) Low magnification images of DAPI-stained nuclei of whole-mount gonads from age-matched wild type and syp-4(tm2713) adult hermaphrodites. The extended transition zone chromosome configuration is observed until late pachytene in the latter. Progression from early to mid-prophase is observed from left to right. (C–H) High magnification images of DAPI-stained nuclei at transition zone (C,F), pachytene (D,G), and diakinesis (E,G) in wild type (C–E) and syp-4(tm2713) mutants (F–H). Bars, 5 µm (B) and 2 µm (C–H).

Mentions: We applied a yeast two-hybrid approach to identify novel proteins functioning in the SC in C. elegans. Specifically, we screened a cDNA library prepared from mixed-stage worms, utilizing SYP-1, SYP-2 and SYP-3 full-length constructs as well as N- and C-terminal truncations as baits (see Materials and Methods). As a result, we identified SYP-4 (encoded by open reading frame H27M09.3) as a protein that interacts with both the full-length and C-terminal truncation constructs of SYP-3. Screens performed with SYP-1 and SYP-2 as baits failed to identify SYP-4 as an interacting protein. In addition, when directly tested for a yeast two-hybrid interaction, SYP-4 failed to interact with the full length, as well as the C- or N-terminal truncation constructs of SYP-1 and SYP-2 (Figure 1, Figure S1). SYP-4 encodes for a 605 amino acid protein. It is predicted to contain three stretches of coiled-coil structure in the region between residues 115 and 410, based on analysis using the COILS program [25]. SYP-4 lacks any other evident structural domains or shared homology with other proteins in C. elegans or other organisms (Figure 2A).


A yeast two-hybrid screen for SYP-3 interactors identifies SYP-4, a component required for synaptonemal complex assembly and chiasma formation in Caenorhabditis elegans meiosis.

Smolikov S, Schild-Prüfert K, Colaiácovo MP - PLoS Genet. (2009)

Extended transition zone chromosome morphology and lack of chiasmata in meiotic prophase I nuclei in syp-4 mutants.(A) Schematic representation of the predicted SYP-4 protein. The region deleted in the tm2713 mutant allele is indicated (codons 28 through 84). Coiled-coil domains, with start and end points identified through the COILS program are shaded in dark gray. The black bar indicates the N-terminal region used for antibody production. (B) Low magnification images of DAPI-stained nuclei of whole-mount gonads from age-matched wild type and syp-4(tm2713) adult hermaphrodites. The extended transition zone chromosome configuration is observed until late pachytene in the latter. Progression from early to mid-prophase is observed from left to right. (C–H) High magnification images of DAPI-stained nuclei at transition zone (C,F), pachytene (D,G), and diakinesis (E,G) in wild type (C–E) and syp-4(tm2713) mutants (F–H). Bars, 5 µm (B) and 2 µm (C–H).
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pgen-1000669-g002: Extended transition zone chromosome morphology and lack of chiasmata in meiotic prophase I nuclei in syp-4 mutants.(A) Schematic representation of the predicted SYP-4 protein. The region deleted in the tm2713 mutant allele is indicated (codons 28 through 84). Coiled-coil domains, with start and end points identified through the COILS program are shaded in dark gray. The black bar indicates the N-terminal region used for antibody production. (B) Low magnification images of DAPI-stained nuclei of whole-mount gonads from age-matched wild type and syp-4(tm2713) adult hermaphrodites. The extended transition zone chromosome configuration is observed until late pachytene in the latter. Progression from early to mid-prophase is observed from left to right. (C–H) High magnification images of DAPI-stained nuclei at transition zone (C,F), pachytene (D,G), and diakinesis (E,G) in wild type (C–E) and syp-4(tm2713) mutants (F–H). Bars, 5 µm (B) and 2 µm (C–H).
Mentions: We applied a yeast two-hybrid approach to identify novel proteins functioning in the SC in C. elegans. Specifically, we screened a cDNA library prepared from mixed-stage worms, utilizing SYP-1, SYP-2 and SYP-3 full-length constructs as well as N- and C-terminal truncations as baits (see Materials and Methods). As a result, we identified SYP-4 (encoded by open reading frame H27M09.3) as a protein that interacts with both the full-length and C-terminal truncation constructs of SYP-3. Screens performed with SYP-1 and SYP-2 as baits failed to identify SYP-4 as an interacting protein. In addition, when directly tested for a yeast two-hybrid interaction, SYP-4 failed to interact with the full length, as well as the C- or N-terminal truncation constructs of SYP-1 and SYP-2 (Figure 1, Figure S1). SYP-4 encodes for a 605 amino acid protein. It is predicted to contain three stretches of coiled-coil structure in the region between residues 115 and 410, based on analysis using the COILS program [25]. SYP-4 lacks any other evident structural domains or shared homology with other proteins in C. elegans or other organisms (Figure 2A).

Bottom Line: SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes.The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality.Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The proper assembly of the synaptonemal complex (SC) between homologs is critical to ensure accurate meiotic chromosome segregation. The SC is a meiotic tripartite structure present from yeast to humans, comprised of proteins assembled along the axes of the chromosomes and central region (CR) proteins that bridge the two chromosome axes. Here we identify SYP-4 as a novel structural component of the SC in Caenorhabditis elegans. SYP-4 interacts in a yeast two-hybrid assay with SYP-3, one of components of the CR of the SC, and is localized at the interface between homologs during meiosis. SYP-4 is essential for the localization of SYP-1, SYP-2, and SYP-3 CR proteins onto chromosomes, thereby playing a crucial role in the stabilization of pairing interactions between homologous chromosomes. In the absence of SYP-4, the levels of recombination intermediates, as indicated by RAD-51 foci, are elevated in mid-prophase nuclei, and crossover recombination events are significantly reduced. The lack of chiasmata observed in syp-4 mutants supports the elevated levels of chromosome nondisjunction manifested in high embryonic lethality. Altogether our findings place SYP-4 as a central player in SC formation and broaden our understanding of the structure of the SC and its assembly.

Show MeSH
Related in: MedlinePlus