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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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Identification of a Spag16L promoter region activated in the presence of SPAG16S.Relative luciferase activity, normalized to PGL3 control promoter plasmid co-transfected with pTarget control vector plasmid. Beas-2B cells were co-transfected with human Spag16L promoter plasmids (corresponding to the indicated regions upstream of the transcription start site) and either pTarget control or SPAG16S/pTarget. All promoter constructs except the −100 bp promoter demonstrated significantly increased activity in the presence of SPAG16S co-transfection. (* p<0.05 compared with pTarget co-transfection for a given promoter).
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pone-0020625-g009: Identification of a Spag16L promoter region activated in the presence of SPAG16S.Relative luciferase activity, normalized to PGL3 control promoter plasmid co-transfected with pTarget control vector plasmid. Beas-2B cells were co-transfected with human Spag16L promoter plasmids (corresponding to the indicated regions upstream of the transcription start site) and either pTarget control or SPAG16S/pTarget. All promoter constructs except the −100 bp promoter demonstrated significantly increased activity in the presence of SPAG16S co-transfection. (* p<0.05 compared with pTarget co-transfection for a given promoter).

Mentions: In order to identify a specific region within the SPAG16L promoter showing increased activity with SPAG16S expression, luciferase plasmid constructs were made with progressively shorter sequences of the SPAG16L proximal promoter, ranging from 2 kb to 100 bp upstream of the transcription start site. Promoters ranging from 2 kb to 200 bp upstream of the transcription start site demonstrated significantly higher activity when co-transfected with a SPAG16S plasmid, but a 100 bp upstream SPAG16L promoter did not show increased activity in the presence of SPAG16S plasmid (Fig. 9).


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)

Identification of a Spag16L promoter region activated in the presence of SPAG16S.Relative luciferase activity, normalized to PGL3 control promoter plasmid co-transfected with pTarget control vector plasmid. Beas-2B cells were co-transfected with human Spag16L promoter plasmids (corresponding to the indicated regions upstream of the transcription start site) and either pTarget control or SPAG16S/pTarget. All promoter constructs except the −100 bp promoter demonstrated significantly increased activity in the presence of SPAG16S co-transfection. (* p<0.05 compared with pTarget co-transfection for a given promoter).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020625-g009: Identification of a Spag16L promoter region activated in the presence of SPAG16S.Relative luciferase activity, normalized to PGL3 control promoter plasmid co-transfected with pTarget control vector plasmid. Beas-2B cells were co-transfected with human Spag16L promoter plasmids (corresponding to the indicated regions upstream of the transcription start site) and either pTarget control or SPAG16S/pTarget. All promoter constructs except the −100 bp promoter demonstrated significantly increased activity in the presence of SPAG16S co-transfection. (* p<0.05 compared with pTarget co-transfection for a given promoter).
Mentions: In order to identify a specific region within the SPAG16L promoter showing increased activity with SPAG16S expression, luciferase plasmid constructs were made with progressively shorter sequences of the SPAG16L proximal promoter, ranging from 2 kb to 100 bp upstream of the transcription start site. Promoters ranging from 2 kb to 200 bp upstream of the transcription start site demonstrated significantly higher activity when co-transfected with a SPAG16S plasmid, but a 100 bp upstream SPAG16L promoter did not show increased activity in the presence of SPAG16S plasmid (Fig. 9).

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