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Spag16, an axonemal central apparatus gene, encodes a male germ cell nuclear speckle protein that regulates SPAG16 mRNA expression.

Nagarkatti-Gude DR, Jaimez R, Henderson SC, Teves ME, Zhang Z, Strauss JF - PLoS ONE (2011)

Bottom Line: Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis.Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors.This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, United States of America.

ABSTRACT
Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the "9+2" axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

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SPAG16S shows enriched nuclear sub-localization.Mixed male germ cells from wild-type or transgenic SPAG16L-KO mice immunolabeled with SPAG16 antibodies (red; N-terminal = SPAG16L only, C-terminal = both isoforms) and nuclear-stained with DAPI (blue). Pre-immune serum for each antibody is included as a negative control.
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pone-0020625-g004: SPAG16S shows enriched nuclear sub-localization.Mixed male germ cells from wild-type or transgenic SPAG16L-KO mice immunolabeled with SPAG16 antibodies (red; N-terminal = SPAG16L only, C-terminal = both isoforms) and nuclear-stained with DAPI (blue). Pre-immune serum for each antibody is included as a negative control.

Mentions: A mixed population of mouse male germ cells was prepared from adult mouse testis, and immunocytochemistry performed to allow for single-cell imaging to compare with immunohistochemistry results. Consistent with previous data, the C-terminal SPAG16 antibody produced the strongest fluorescence in discrete sub-nuclear, non-nucleolar structures, approximately 2–6 per cell (Fig. 4). The N-terminal SPAG16 antibody, recognizing SPAG16L only, produced an exclusively cytoplasmic signal. Germ cells from SPAG16L-KO mice [9] were isolated and immunolabeled as well, demonstrating the specificity of the antibodies (Fig. 4). As in wild-type germ cells, the C-terminal SPAG16 antibody produced sub-nuclear immunolabelling, which we interpret to represent SPAG16S, while the N-terminal antibody produced no signal, as expected following the deletion of SPAG16L.


Spag16, an axonemal central apparatus gene, encodes a male germ cell nuclear speckle protein that regulates SPAG16 mRNA expression.

Nagarkatti-Gude DR, Jaimez R, Henderson SC, Teves ME, Zhang Z, Strauss JF - PLoS ONE (2011)

SPAG16S shows enriched nuclear sub-localization.Mixed male germ cells from wild-type or transgenic SPAG16L-KO mice immunolabeled with SPAG16 antibodies (red; N-terminal = SPAG16L only, C-terminal = both isoforms) and nuclear-stained with DAPI (blue). Pre-immune serum for each antibody is included as a negative control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105110&req=5

pone-0020625-g004: SPAG16S shows enriched nuclear sub-localization.Mixed male germ cells from wild-type or transgenic SPAG16L-KO mice immunolabeled with SPAG16 antibodies (red; N-terminal = SPAG16L only, C-terminal = both isoforms) and nuclear-stained with DAPI (blue). Pre-immune serum for each antibody is included as a negative control.
Mentions: A mixed population of mouse male germ cells was prepared from adult mouse testis, and immunocytochemistry performed to allow for single-cell imaging to compare with immunohistochemistry results. Consistent with previous data, the C-terminal SPAG16 antibody produced the strongest fluorescence in discrete sub-nuclear, non-nucleolar structures, approximately 2–6 per cell (Fig. 4). The N-terminal SPAG16 antibody, recognizing SPAG16L only, produced an exclusively cytoplasmic signal. Germ cells from SPAG16L-KO mice [9] were isolated and immunolabeled as well, demonstrating the specificity of the antibodies (Fig. 4). As in wild-type germ cells, the C-terminal SPAG16 antibody produced sub-nuclear immunolabelling, which we interpret to represent SPAG16S, while the N-terminal antibody produced no signal, as expected following the deletion of SPAG16L.

Bottom Line: Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis.Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors.This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia, United States of America.

ABSTRACT
Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the "9+2" axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.

Show MeSH
Related in: MedlinePlus