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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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SPAG16L is in the cytoplasm of male germ cells, SPAG16S is in both the nucleus and the cytoplasm.(A) Cytoplasmic and nuclear fractions of adult mouse testis probed by Western blot for SPAG16 (C-terminal antibody recognizing both isoforms) or markers of cytoplasm (α-tubulin) or nucleus (Lamin B). (B) Sections of adult mouse testis immunolabeled with SPAG16 C-terminal antibody or pre-immune serum (negative control). Arrows indicate sample nuclear regions of heightened SPAG16 antibody immunoreactivity.
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pone-0020625-g003: SPAG16L is in the cytoplasm of male germ cells, SPAG16S is in both the nucleus and the cytoplasm.(A) Cytoplasmic and nuclear fractions of adult mouse testis probed by Western blot for SPAG16 (C-terminal antibody recognizing both isoforms) or markers of cytoplasm (α-tubulin) or nucleus (Lamin B). (B) Sections of adult mouse testis immunolabeled with SPAG16 C-terminal antibody or pre-immune serum (negative control). Arrows indicate sample nuclear regions of heightened SPAG16 antibody immunoreactivity.

Mentions: Cytoplasmic and nuclear fractions of adult mouse testis were isolated, and equivalent amounts of protein from each were probed by Western blot using the C-terminal SPAG16 antibody that recognizes both isoforms. SPAG16L was detected abundantly in the cytoplasm, while SPAG16S was detected in both cytoplasm and nucleus (Fig. 3A). The cytoplasmic localization of SPAG16L is consistent with its identified role as a structural component of the “9+2” axoneme [6], [16], [17]. In order to further characterize the sub-cellular localization of SPAG16S, immunohistochemistry was performed on tissue slices from adult mouse testis. Using the C-terminal SPAG16 antibody, the strongest signal was detected from discrete structures within the nucleus (Fig. 3B, see arrows). Protein expression was most clearly visualized approximately halfway through spermatogenesis, at the round spermatid stage.


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)

SPAG16L is in the cytoplasm of male germ cells, SPAG16S is in both the nucleus and the cytoplasm.(A) Cytoplasmic and nuclear fractions of adult mouse testis probed by Western blot for SPAG16 (C-terminal antibody recognizing both isoforms) or markers of cytoplasm (α-tubulin) or nucleus (Lamin B). (B) Sections of adult mouse testis immunolabeled with SPAG16 C-terminal antibody or pre-immune serum (negative control). Arrows indicate sample nuclear regions of heightened SPAG16 antibody immunoreactivity.
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Related In: Results  -  Collection

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

pone-0020625-g003: SPAG16L is in the cytoplasm of male germ cells, SPAG16S is in both the nucleus and the cytoplasm.(A) Cytoplasmic and nuclear fractions of adult mouse testis probed by Western blot for SPAG16 (C-terminal antibody recognizing both isoforms) or markers of cytoplasm (α-tubulin) or nucleus (Lamin B). (B) Sections of adult mouse testis immunolabeled with SPAG16 C-terminal antibody or pre-immune serum (negative control). Arrows indicate sample nuclear regions of heightened SPAG16 antibody immunoreactivity.
Mentions: Cytoplasmic and nuclear fractions of adult mouse testis were isolated, and equivalent amounts of protein from each were probed by Western blot using the C-terminal SPAG16 antibody that recognizes both isoforms. SPAG16L was detected abundantly in the cytoplasm, while SPAG16S was detected in both cytoplasm and nucleus (Fig. 3A). The cytoplasmic localization of SPAG16L is consistent with its identified role as a structural component of the “9+2” axoneme [6], [16], [17]. In order to further characterize the sub-cellular localization of SPAG16S, immunohistochemistry was performed on tissue slices from adult mouse testis. Using the C-terminal SPAG16 antibody, the strongest signal was detected from discrete structures within the nucleus (Fig. 3B, see arrows). Protein expression was most clearly visualized approximately halfway through spermatogenesis, at the round spermatid stage.

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