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An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Yang Z, Gallicano GI, Yu QC, Fuchs E - J. Cell Biol. (1997)

Bottom Line: In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes.Cell.Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

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Northern blot and  RNase protection analyses.  Northern blots of mouse and  human RNAs were purchased from Clontech. Blots  contained 2 μg polyA+  mRNAs, which were hybridized with a radiolabeled  probe corresponding to a  576-bp fragment shared by  BSN1a and BSN1b. As control, a radiolabeled probe  corresponding to a 1-kb fragment of glyceraldehyde dehydrogenase was used. Note  the presence of a 4,600-nucleotide band, predicted for the  bona fide BSN1a and BSN1b RNAs, in testes samples (TS) from both mouse (m) and human (h); note the presence of an additional  3,200-nucleotide band in human testis. PB, peripheral blood; CO, colon; SI, small intestine; OV, ovary; TS, testis; PS, prostate; TH, thymus; SL, spleen. (B) RNAs were isolated from the testes of mice at 2 wk, 5 wk, and adult postnatally. RNAs were hybridized with radiolabeled mouse cRNas generated to either (a) a 576-nucleotide sequence common to BSN1a and BSN1b or (b) a 359-nucleotide actin sequence. After hybridization, RNase treatment was performed as described by Faus et al. (1994). Duplexes were resolved by PAGE, and  the gel was exposed to x-ray film overnight. Note the presence of 487- and 250-nucleotide bands expected for BSN1a/BSN1b and actin  RNAs, respectively, after hybridization with the probes and RNase digestion.
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Figure 4: Northern blot and RNase protection analyses. Northern blots of mouse and human RNAs were purchased from Clontech. Blots contained 2 μg polyA+ mRNAs, which were hybridized with a radiolabeled probe corresponding to a 576-bp fragment shared by BSN1a and BSN1b. As control, a radiolabeled probe corresponding to a 1-kb fragment of glyceraldehyde dehydrogenase was used. Note the presence of a 4,600-nucleotide band, predicted for the bona fide BSN1a and BSN1b RNAs, in testes samples (TS) from both mouse (m) and human (h); note the presence of an additional 3,200-nucleotide band in human testis. PB, peripheral blood; CO, colon; SI, small intestine; OV, ovary; TS, testis; PS, prostate; TH, thymus; SL, spleen. (B) RNAs were isolated from the testes of mice at 2 wk, 5 wk, and adult postnatally. RNAs were hybridized with radiolabeled mouse cRNas generated to either (a) a 576-nucleotide sequence common to BSN1a and BSN1b or (b) a 359-nucleotide actin sequence. After hybridization, RNase treatment was performed as described by Faus et al. (1994). Duplexes were resolved by PAGE, and the gel was exposed to x-ray film overnight. Note the presence of 487- and 250-nucleotide bands expected for BSN1a/BSN1b and actin RNAs, respectively, after hybridization with the probes and RNase digestion.

Mentions: To examine BSN expression in testis in more detail, we first conducted Northern blot analysis. As shown in Fig. 4 A, a single RNA band of ∼4,600 nucleotides was obtained from mouse testis. This band was comparable in size to that seen in human keratinocyte mRNA preparations (Tseng and Green, 1994), and it corresponded to the size expected for BSN1a and BSN1b mRNAs, which have a long 3′ untranslated sequence. For human testis, two bands were detected in approximately comparable levels: one was an ∼4,600-nucleotide band, as expected, and the other was an ∼3,200-nucleotide band. This smaller band is large enough to encode full-length basonuclin, although our focus for the remainder of the study was on mouse, and we have not pursued the identity of this smaller band in human testis.


An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Yang Z, Gallicano GI, Yu QC, Fuchs E - J. Cell Biol. (1997)

Northern blot and  RNase protection analyses.  Northern blots of mouse and  human RNAs were purchased from Clontech. Blots  contained 2 μg polyA+  mRNAs, which were hybridized with a radiolabeled  probe corresponding to a  576-bp fragment shared by  BSN1a and BSN1b. As control, a radiolabeled probe  corresponding to a 1-kb fragment of glyceraldehyde dehydrogenase was used. Note  the presence of a 4,600-nucleotide band, predicted for the  bona fide BSN1a and BSN1b RNAs, in testes samples (TS) from both mouse (m) and human (h); note the presence of an additional  3,200-nucleotide band in human testis. PB, peripheral blood; CO, colon; SI, small intestine; OV, ovary; TS, testis; PS, prostate; TH, thymus; SL, spleen. (B) RNAs were isolated from the testes of mice at 2 wk, 5 wk, and adult postnatally. RNAs were hybridized with radiolabeled mouse cRNas generated to either (a) a 576-nucleotide sequence common to BSN1a and BSN1b or (b) a 359-nucleotide actin sequence. After hybridization, RNase treatment was performed as described by Faus et al. (1994). Duplexes were resolved by PAGE, and  the gel was exposed to x-ray film overnight. Note the presence of 487- and 250-nucleotide bands expected for BSN1a/BSN1b and actin  RNAs, respectively, after hybridization with the probes and RNase digestion.
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Related In: Results  -  Collection

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Figure 4: Northern blot and RNase protection analyses. Northern blots of mouse and human RNAs were purchased from Clontech. Blots contained 2 μg polyA+ mRNAs, which were hybridized with a radiolabeled probe corresponding to a 576-bp fragment shared by BSN1a and BSN1b. As control, a radiolabeled probe corresponding to a 1-kb fragment of glyceraldehyde dehydrogenase was used. Note the presence of a 4,600-nucleotide band, predicted for the bona fide BSN1a and BSN1b RNAs, in testes samples (TS) from both mouse (m) and human (h); note the presence of an additional 3,200-nucleotide band in human testis. PB, peripheral blood; CO, colon; SI, small intestine; OV, ovary; TS, testis; PS, prostate; TH, thymus; SL, spleen. (B) RNAs were isolated from the testes of mice at 2 wk, 5 wk, and adult postnatally. RNAs were hybridized with radiolabeled mouse cRNas generated to either (a) a 576-nucleotide sequence common to BSN1a and BSN1b or (b) a 359-nucleotide actin sequence. After hybridization, RNase treatment was performed as described by Faus et al. (1994). Duplexes were resolved by PAGE, and the gel was exposed to x-ray film overnight. Note the presence of 487- and 250-nucleotide bands expected for BSN1a/BSN1b and actin RNAs, respectively, after hybridization with the probes and RNase digestion.
Mentions: To examine BSN expression in testis in more detail, we first conducted Northern blot analysis. As shown in Fig. 4 A, a single RNA band of ∼4,600 nucleotides was obtained from mouse testis. This band was comparable in size to that seen in human keratinocyte mRNA preparations (Tseng and Green, 1994), and it corresponded to the size expected for BSN1a and BSN1b mRNAs, which have a long 3′ untranslated sequence. For human testis, two bands were detected in approximately comparable levels: one was an ∼4,600-nucleotide band, as expected, and the other was an ∼3,200-nucleotide band. This smaller band is large enough to encode full-length basonuclin, although our focus for the remainder of the study was on mouse, and we have not pursued the identity of this smaller band in human testis.

Bottom Line: In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes.Cell.Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Show MeSH
Related in: MedlinePlus