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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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The murine Spag16 gene encodes two transcripts.Spag16L is expressed in all tissues with ciliated cells, while Spag16S is expressed only in testis. (A) 5′ RACE was performed with a primer as indicated. (B) Products of 5′ RACE separated on 1% agarose gel. (C) Exon map of Spag16 transcripts, unfilled box indicating untranslated exon 10a present only in Spag16S. Arrows indicate primers used to amplify specifically Spag16L or Spag16S message. (D) Specific primer sets were used as indicated for PCR amplification of cDNA from adult mouse tissues: Testis (T), Brain (B), Lungs (Lu), Oviduct (Ov), Heart (H).
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pone-0020625-g001: The murine Spag16 gene encodes two transcripts.Spag16L is expressed in all tissues with ciliated cells, while Spag16S is expressed only in testis. (A) 5′ RACE was performed with a primer as indicated. (B) Products of 5′ RACE separated on 1% agarose gel. (C) Exon map of Spag16 transcripts, unfilled box indicating untranslated exon 10a present only in Spag16S. Arrows indicate primers used to amplify specifically Spag16L or Spag16S message. (D) Specific primer sets were used as indicated for PCR amplification of cDNA from adult mouse tissues: Testis (T), Brain (B), Lungs (Lu), Oviduct (Ov), Heart (H).

Mentions: To identify the 5′ UTR of Spag16S mRNA, 5′ RACE was performed with a primer located close to the 3′ end of mouse Spag16L mRNA (Fig. 1A). Two PCR products were amplified (Fig. 1B), and each one was cloned into the pCR2.1 Topo TA vector (Invitrogen). 10 clones of each PCR product were sequenced after vector insertion, demonstrating that Spag16S sequence is identical to that of Spag16L exons 11–17, with the addition of a 5′ untranslated exon, not found in Spag16L, named exon 10a (Fig. 1C). This exon 10a is located in the middle of intron 10 of the Spag16 gene, approximately 50 kb from Spag16L exon 10 and 50 kb from Spag16L exon 11. Sequencing results demonstrated multiple potential transcription start sites for Spag16S transcription; the exon is situated in a TC-rich locus that lacks a standard TATA box (Figure S1).


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)

The murine Spag16 gene encodes two transcripts.Spag16L is expressed in all tissues with ciliated cells, while Spag16S is expressed only in testis. (A) 5′ RACE was performed with a primer as indicated. (B) Products of 5′ RACE separated on 1% agarose gel. (C) Exon map of Spag16 transcripts, unfilled box indicating untranslated exon 10a present only in Spag16S. Arrows indicate primers used to amplify specifically Spag16L or Spag16S message. (D) Specific primer sets were used as indicated for PCR amplification of cDNA from adult mouse tissues: Testis (T), Brain (B), Lungs (Lu), Oviduct (Ov), Heart (H).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105110&req=5

pone-0020625-g001: The murine Spag16 gene encodes two transcripts.Spag16L is expressed in all tissues with ciliated cells, while Spag16S is expressed only in testis. (A) 5′ RACE was performed with a primer as indicated. (B) Products of 5′ RACE separated on 1% agarose gel. (C) Exon map of Spag16 transcripts, unfilled box indicating untranslated exon 10a present only in Spag16S. Arrows indicate primers used to amplify specifically Spag16L or Spag16S message. (D) Specific primer sets were used as indicated for PCR amplification of cDNA from adult mouse tissues: Testis (T), Brain (B), Lungs (Lu), Oviduct (Ov), Heart (H).
Mentions: To identify the 5′ UTR of Spag16S mRNA, 5′ RACE was performed with a primer located close to the 3′ end of mouse Spag16L mRNA (Fig. 1A). Two PCR products were amplified (Fig. 1B), and each one was cloned into the pCR2.1 Topo TA vector (Invitrogen). 10 clones of each PCR product were sequenced after vector insertion, demonstrating that Spag16S sequence is identical to that of Spag16L exons 11–17, with the addition of a 5′ untranslated exon, not found in Spag16L, named exon 10a (Fig. 1C). This exon 10a is located in the middle of intron 10 of the Spag16 gene, approximately 50 kb from Spag16L exon 10 and 50 kb from Spag16L exon 11. Sequencing results demonstrated multiple potential transcription start sites for Spag16S transcription; the exon is situated in a TC-rich locus that lacks a standard TATA box (Figure S1).

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