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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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SPAG16 isoforms have identical conserved domains.SPAG16S appears before SPAG16L during the first wave of mouse spermatogenesis. (A) Alignment and conserved domain analysis of SPAG16L and SPAG16S proteins. RNA (B) and protein (C) were isolated from mouse testis at the indicated day (bottom row) after birth. (B) Specific primer sets were used to probe gene expression by PCR of cDNA. (C) Protein samples were separated by SDS-PAGE and probed by Western blotting with a C-terminal SPAG16 antibody that recognizes both SPAG16L (71 kDa band) and SPAG16S (35 kDa band). Additional bands are not specific.
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pone-0020625-g002: SPAG16 isoforms have identical conserved domains.SPAG16S appears before SPAG16L during the first wave of mouse spermatogenesis. (A) Alignment and conserved domain analysis of SPAG16L and SPAG16S proteins. RNA (B) and protein (C) were isolated from mouse testis at the indicated day (bottom row) after birth. (B) Specific primer sets were used to probe gene expression by PCR of cDNA. (C) Protein samples were separated by SDS-PAGE and probed by Western blotting with a C-terminal SPAG16 antibody that recognizes both SPAG16L (71 kDa band) and SPAG16S (35 kDa band). Additional bands are not specific.

Mentions: RNA and protein were isolated from mouse testis at 6, 8, 12, 16, 20, 30, and 42 days after birth. cDNA was generated by RT-PCR, and testis extracts were probed by PCR for Spag16 isoform expression using specific primers. Spag16S mRNA was detected at day 16, whereas Spag16L mRNA was detected later, at day 20 (Fig. 2B). Western blotting was performed as well, using a polyclonal antibody that recognizes both isoforms of SPAG16 [8]. Consistent with PCR results, SPAG16S protein was detected at day 16, while SPAG16L was detected at day 20 (Fig. 2C). Additionally, both isoforms appeared to be up-regulated at days 30 and 42, consistent with the end of the first wave of spermatogenesis. The immunoreactive bands other than 71 kDa SPAG16L and 35 kDA SPAG16S seen on days 20, 30 and 42 may represent post-translational processing of SPAG16, including proteolytic cleavage of SPAG16L and phosphorylation or other modifications [15].


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)

SPAG16 isoforms have identical conserved domains.SPAG16S appears before SPAG16L during the first wave of mouse spermatogenesis. (A) Alignment and conserved domain analysis of SPAG16L and SPAG16S proteins. RNA (B) and protein (C) were isolated from mouse testis at the indicated day (bottom row) after birth. (B) Specific primer sets were used to probe gene expression by PCR of cDNA. (C) Protein samples were separated by SDS-PAGE and probed by Western blotting with a C-terminal SPAG16 antibody that recognizes both SPAG16L (71 kDa band) and SPAG16S (35 kDa band). Additional bands are not specific.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105110&req=5

pone-0020625-g002: SPAG16 isoforms have identical conserved domains.SPAG16S appears before SPAG16L during the first wave of mouse spermatogenesis. (A) Alignment and conserved domain analysis of SPAG16L and SPAG16S proteins. RNA (B) and protein (C) were isolated from mouse testis at the indicated day (bottom row) after birth. (B) Specific primer sets were used to probe gene expression by PCR of cDNA. (C) Protein samples were separated by SDS-PAGE and probed by Western blotting with a C-terminal SPAG16 antibody that recognizes both SPAG16L (71 kDa band) and SPAG16S (35 kDa band). Additional bands are not specific.
Mentions: RNA and protein were isolated from mouse testis at 6, 8, 12, 16, 20, 30, and 42 days after birth. cDNA was generated by RT-PCR, and testis extracts were probed by PCR for Spag16 isoform expression using specific primers. Spag16S mRNA was detected at day 16, whereas Spag16L mRNA was detected later, at day 20 (Fig. 2B). Western blotting was performed as well, using a polyclonal antibody that recognizes both isoforms of SPAG16 [8]. Consistent with PCR results, SPAG16S protein was detected at day 16, while SPAG16L was detected at day 20 (Fig. 2C). Additionally, both isoforms appeared to be up-regulated at days 30 and 42, consistent with the end of the first wave of spermatogenesis. The immunoreactive bands other than 71 kDa SPAG16L and 35 kDA SPAG16S seen on days 20, 30 and 42 may represent post-translational processing of SPAG16, including proteolytic cleavage of SPAG16L and phosphorylation or other modifications [15].

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