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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 stimulates Spag16L mRNA and SPAG16L protein expression in BEAS-2B cells.Analysis of Spag16L mRNA expression by real-time PCR in BEAS-2B cells stably expressing SPAG16S (A) or transduced with Ad/SPAG16S (B). (C) Adenovirus-transduced BEAS-2B cells immunolabeled with a C-terminal SPAG16 antibody. (D) Analysis of SPAG16L protein expression by Western blotting in BEAS-2B cells infected by Ad/SPAG16S. This panel demonstrates two independent experiments. (E) Relative intensity of SPAG16L signal in panel D, normalized to Actin loading control for each sample (relative Actin signal for Ad/Control = 47.5%, for Ad/SPAG16S = 52.5%) (* p<0.05 compared with pTarget or Ad/Control).
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pone-0020625-g007: SPAG16S stimulates Spag16L mRNA and SPAG16L protein expression in BEAS-2B cells.Analysis of Spag16L mRNA expression by real-time PCR in BEAS-2B cells stably expressing SPAG16S (A) or transduced with Ad/SPAG16S (B). (C) Adenovirus-transduced BEAS-2B cells immunolabeled with a C-terminal SPAG16 antibody. (D) Analysis of SPAG16L protein expression by Western blotting in BEAS-2B cells infected by Ad/SPAG16S. This panel demonstrates two independent experiments. (E) Relative intensity of SPAG16L signal in panel D, normalized to Actin loading control for each sample (relative Actin signal for Ad/Control = 47.5%, for Ad/SPAG16S = 52.5%) (* p<0.05 compared with pTarget or Ad/Control).

Mentions: SPAG16S was also shown to induce SPAG16L expression in BEAS-2B human bronchial epithelial cells. Thiscell line does not express SPAG16L at levels detectable by qPCR or Western blotting (data not shown). Expression of SPAG16S was induced in cultured BEAS-2B cells by plasmid (Fig. 7A) or adenovirus (Fig. 7B), and primers specific for human SPAG16L were used to measure transcript levels, which were normalized to 18S rRNA. Immunocytochemistry performed with the C-terminal SPAG16 antibody confirms that SPAG16 proteins are expressed in the transduced cells, but not in cells exposed to a control adenovirus (Fig. 7C). Following adenoviral transduction, protein was also isolated from cultured BEAS-2B cells, and SPAG16L was demonstrated by Western blot to be present in cells transduced with SPAG16S adenovirus, but not cells transduced with a control adenovirus (Fig. 7D).


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 stimulates Spag16L mRNA and SPAG16L protein expression in BEAS-2B cells.Analysis of Spag16L mRNA expression by real-time PCR in BEAS-2B cells stably expressing SPAG16S (A) or transduced with Ad/SPAG16S (B). (C) Adenovirus-transduced BEAS-2B cells immunolabeled with a C-terminal SPAG16 antibody. (D) Analysis of SPAG16L protein expression by Western blotting in BEAS-2B cells infected by Ad/SPAG16S. This panel demonstrates two independent experiments. (E) Relative intensity of SPAG16L signal in panel D, normalized to Actin loading control for each sample (relative Actin signal for Ad/Control = 47.5%, for Ad/SPAG16S = 52.5%) (* p<0.05 compared with pTarget or Ad/Control).
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getmorefigures.php?uid=PMC3105110&req=5

pone-0020625-g007: SPAG16S stimulates Spag16L mRNA and SPAG16L protein expression in BEAS-2B cells.Analysis of Spag16L mRNA expression by real-time PCR in BEAS-2B cells stably expressing SPAG16S (A) or transduced with Ad/SPAG16S (B). (C) Adenovirus-transduced BEAS-2B cells immunolabeled with a C-terminal SPAG16 antibody. (D) Analysis of SPAG16L protein expression by Western blotting in BEAS-2B cells infected by Ad/SPAG16S. This panel demonstrates two independent experiments. (E) Relative intensity of SPAG16L signal in panel D, normalized to Actin loading control for each sample (relative Actin signal for Ad/Control = 47.5%, for Ad/SPAG16S = 52.5%) (* p<0.05 compared with pTarget or Ad/Control).
Mentions: SPAG16S was also shown to induce SPAG16L expression in BEAS-2B human bronchial epithelial cells. Thiscell line does not express SPAG16L at levels detectable by qPCR or Western blotting (data not shown). Expression of SPAG16S was induced in cultured BEAS-2B cells by plasmid (Fig. 7A) or adenovirus (Fig. 7B), and primers specific for human SPAG16L were used to measure transcript levels, which were normalized to 18S rRNA. Immunocytochemistry performed with the C-terminal SPAG16 antibody confirms that SPAG16 proteins are expressed in the transduced cells, but not in cells exposed to a control adenovirus (Fig. 7C). Following adenoviral transduction, protein was also isolated from cultured BEAS-2B cells, and SPAG16L was demonstrated by Western blot to be present in cells transduced with SPAG16S adenovirus, but not cells transduced with a control adenovirus (Fig. 7D).

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