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The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles.

Leung CL, Zheng M, Prater SM, Liem RK - J. Cell Biol. (2001)

Bottom Line: We have analyzed the BPAG1 locus in detail and found that it encodes different interaction domains that are combined in tissue-specific manners.BPAG1-a is composed of the ABD, the plakin domain, the SR-containing rod domain, and the MTBD.BPAG1-b is highly expressed in muscles, and has extra exons encoding a second IFBD between the plakin and SR-containing rod domains of BPAG1-a.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

ABSTRACT
Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family with cytoskeletal linker properties. Mutations in BPAG1 cause sensory neuron degeneration and skin fragility in mice. We have analyzed the BPAG1 locus in detail and found that it encodes different interaction domains that are combined in tissue-specific manners. These domains include an actin-binding domain (ABD), a plakin domain, a coiled coil (CC) rod domain, two different potential intermediate filament-binding domains (IFBDs), a spectrin repeat (SR)-containing rod domain, and a microtubule-binding domain (MTBD). There are at least three major forms of BPAG1: BPAG1-e (302 kD), BPAG1-a (615 kD), and BPAG1-b (834 kD). BPAG1-e has been described previously and consists of the plakin domain, the CC rod domain, and the first IFBD. It is the primary epidermal BPAG1 isoform, and its absence that is the likely cause of skin fragility in mutant mice. BPAG1-a is the major isoform in the nervous system and a homologue of the microtubule actin cross-linking factor, MACF. BPAG1-a is composed of the ABD, the plakin domain, the SR-containing rod domain, and the MTBD. The absence of BPAG1-a is the likely cause of sensory neurodegeneration in mutant mice. BPAG1-b is highly expressed in muscles, and has extra exons encoding a second IFBD between the plakin and SR-containing rod domains of BPAG1-a.

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Analyses of BPAG1 mRNAs. (A) A schematic diagram showing the structure of the BPAG1 gene. Bold gray lines underneath individual domains represent the probes used for Northern blot (NB) analysis and in situ hybridizations. The positions of riboprobes (bold lines in black) used for RPA and oligonucleotide primers (arrows) used for RT-PCR analyses are illustrated. The names of the riboprobes corresponded to the BPAG1 structural domains that the riboprobes recognized. The primer sets were named after the BPAG1 isoforms that they amplified. (B) Northern blot analysis of BPAG1 isoforms. The positions of the 17-kb brain MACF mRNA and the 2-kb RNA standard are marked. The β-actin probe also detected other actin isoforms. (C) RPA analysis of BPAG1 isoforms. BPAG1 isoforms that contained the IFBD1 domain were only detected in skin. BPAG1-b transcripts that could be protected by IFBD2 probe were found in large amounts in the heart and small amounts in the brain. The SR-containing rod domain probe that protected both BPAG1-a and BPAG1-b mRNAs gave strong signals in brain and heart and weak signals in skin. Marker standards (100, 200, and 300 bp) are indicated on the left of each panel. (D) RT-PCR analyses of BPAG1 isoforms. The highest amounts of BPAG1-a and BPAG1-b were observed in brain and heart, respectively. BPAG1-e mRNAs were only found in the skin, whereas no BPAG1-e/n mRNAs were detected in the brain or the heart with these RT-PCR settings.
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fig3: Analyses of BPAG1 mRNAs. (A) A schematic diagram showing the structure of the BPAG1 gene. Bold gray lines underneath individual domains represent the probes used for Northern blot (NB) analysis and in situ hybridizations. The positions of riboprobes (bold lines in black) used for RPA and oligonucleotide primers (arrows) used for RT-PCR analyses are illustrated. The names of the riboprobes corresponded to the BPAG1 structural domains that the riboprobes recognized. The primer sets were named after the BPAG1 isoforms that they amplified. (B) Northern blot analysis of BPAG1 isoforms. The positions of the 17-kb brain MACF mRNA and the 2-kb RNA standard are marked. The β-actin probe also detected other actin isoforms. (C) RPA analysis of BPAG1 isoforms. BPAG1 isoforms that contained the IFBD1 domain were only detected in skin. BPAG1-b transcripts that could be protected by IFBD2 probe were found in large amounts in the heart and small amounts in the brain. The SR-containing rod domain probe that protected both BPAG1-a and BPAG1-b mRNAs gave strong signals in brain and heart and weak signals in skin. Marker standards (100, 200, and 300 bp) are indicated on the left of each panel. (D) RT-PCR analyses of BPAG1 isoforms. The highest amounts of BPAG1-a and BPAG1-b were observed in brain and heart, respectively. BPAG1-e mRNAs were only found in the skin, whereas no BPAG1-e/n mRNAs were detected in the brain or the heart with these RT-PCR settings.

Mentions: We prepared probes against different domains of BPAG1 isoforms for Northern blot analysis. Three different hybridization patterns were obtained: the pattern in the brain was identical to the spinal cord, the pattern in the heart was identical to the skeletal muscles, and the pattern in the skin was unique. BPAG1-a mRNA was the only BPAG1 mRNA detected in the brain and the spinal cord (Fig. 3 B and unpublished data). Probes specific for the CC rod domain and IFBD1 failed to recognize any mRNAs in the brain and spinal cord, indicating that BPAG1-n is not a major isoform in the central nervous system. The previously reported BPAG1-n mRNAs in the nervous system are likely due to crosshybridization of BPAG1-a with the BPAG1-n probes, as the probes used in these studies were against the ABD and the plakin domain, common to BPAG1-a and BPAG1-n (Bernier et al., 1995a, 1995b, 1998; Brown et al., 1995; Yang et al., 1996; Dalpe et al., 1998). In the heart, BPAG1-b was the predominant BPAG1 isoform. The mRNA of BPAG1-b was labeled with the IFBD2 probe and was larger than the mRNA of BPAG1-a (Fig. 3 B). These results are consistent with previous studies that showed a larger BPAG1 message in skeletal muscles and the heart with probes specific for the 5′ portions of BPAG1-n (Bernier et al., 1995b; Dalpe et al., 1999). Also in agreement with other studies (Yang et al., 1996; Dalpe et al., 1998), the shorter BPAG1-e transcripts were detected in the skin.


The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles.

Leung CL, Zheng M, Prater SM, Liem RK - J. Cell Biol. (2001)

Analyses of BPAG1 mRNAs. (A) A schematic diagram showing the structure of the BPAG1 gene. Bold gray lines underneath individual domains represent the probes used for Northern blot (NB) analysis and in situ hybridizations. The positions of riboprobes (bold lines in black) used for RPA and oligonucleotide primers (arrows) used for RT-PCR analyses are illustrated. The names of the riboprobes corresponded to the BPAG1 structural domains that the riboprobes recognized. The primer sets were named after the BPAG1 isoforms that they amplified. (B) Northern blot analysis of BPAG1 isoforms. The positions of the 17-kb brain MACF mRNA and the 2-kb RNA standard are marked. The β-actin probe also detected other actin isoforms. (C) RPA analysis of BPAG1 isoforms. BPAG1 isoforms that contained the IFBD1 domain were only detected in skin. BPAG1-b transcripts that could be protected by IFBD2 probe were found in large amounts in the heart and small amounts in the brain. The SR-containing rod domain probe that protected both BPAG1-a and BPAG1-b mRNAs gave strong signals in brain and heart and weak signals in skin. Marker standards (100, 200, and 300 bp) are indicated on the left of each panel. (D) RT-PCR analyses of BPAG1 isoforms. The highest amounts of BPAG1-a and BPAG1-b were observed in brain and heart, respectively. BPAG1-e mRNAs were only found in the skin, whereas no BPAG1-e/n mRNAs were detected in the brain or the heart with these RT-PCR settings.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196450&req=5

fig3: Analyses of BPAG1 mRNAs. (A) A schematic diagram showing the structure of the BPAG1 gene. Bold gray lines underneath individual domains represent the probes used for Northern blot (NB) analysis and in situ hybridizations. The positions of riboprobes (bold lines in black) used for RPA and oligonucleotide primers (arrows) used for RT-PCR analyses are illustrated. The names of the riboprobes corresponded to the BPAG1 structural domains that the riboprobes recognized. The primer sets were named after the BPAG1 isoforms that they amplified. (B) Northern blot analysis of BPAG1 isoforms. The positions of the 17-kb brain MACF mRNA and the 2-kb RNA standard are marked. The β-actin probe also detected other actin isoforms. (C) RPA analysis of BPAG1 isoforms. BPAG1 isoforms that contained the IFBD1 domain were only detected in skin. BPAG1-b transcripts that could be protected by IFBD2 probe were found in large amounts in the heart and small amounts in the brain. The SR-containing rod domain probe that protected both BPAG1-a and BPAG1-b mRNAs gave strong signals in brain and heart and weak signals in skin. Marker standards (100, 200, and 300 bp) are indicated on the left of each panel. (D) RT-PCR analyses of BPAG1 isoforms. The highest amounts of BPAG1-a and BPAG1-b were observed in brain and heart, respectively. BPAG1-e mRNAs were only found in the skin, whereas no BPAG1-e/n mRNAs were detected in the brain or the heart with these RT-PCR settings.
Mentions: We prepared probes against different domains of BPAG1 isoforms for Northern blot analysis. Three different hybridization patterns were obtained: the pattern in the brain was identical to the spinal cord, the pattern in the heart was identical to the skeletal muscles, and the pattern in the skin was unique. BPAG1-a mRNA was the only BPAG1 mRNA detected in the brain and the spinal cord (Fig. 3 B and unpublished data). Probes specific for the CC rod domain and IFBD1 failed to recognize any mRNAs in the brain and spinal cord, indicating that BPAG1-n is not a major isoform in the central nervous system. The previously reported BPAG1-n mRNAs in the nervous system are likely due to crosshybridization of BPAG1-a with the BPAG1-n probes, as the probes used in these studies were against the ABD and the plakin domain, common to BPAG1-a and BPAG1-n (Bernier et al., 1995a, 1995b, 1998; Brown et al., 1995; Yang et al., 1996; Dalpe et al., 1998). In the heart, BPAG1-b was the predominant BPAG1 isoform. The mRNA of BPAG1-b was labeled with the IFBD2 probe and was larger than the mRNA of BPAG1-a (Fig. 3 B). These results are consistent with previous studies that showed a larger BPAG1 message in skeletal muscles and the heart with probes specific for the 5′ portions of BPAG1-n (Bernier et al., 1995b; Dalpe et al., 1999). Also in agreement with other studies (Yang et al., 1996; Dalpe et al., 1998), the shorter BPAG1-e transcripts were detected in the skin.

Bottom Line: We have analyzed the BPAG1 locus in detail and found that it encodes different interaction domains that are combined in tissue-specific manners.BPAG1-a is composed of the ABD, the plakin domain, the SR-containing rod domain, and the MTBD.BPAG1-b is highly expressed in muscles, and has extra exons encoding a second IFBD between the plakin and SR-containing rod domains of BPAG1-a.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

ABSTRACT
Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family with cytoskeletal linker properties. Mutations in BPAG1 cause sensory neuron degeneration and skin fragility in mice. We have analyzed the BPAG1 locus in detail and found that it encodes different interaction domains that are combined in tissue-specific manners. These domains include an actin-binding domain (ABD), a plakin domain, a coiled coil (CC) rod domain, two different potential intermediate filament-binding domains (IFBDs), a spectrin repeat (SR)-containing rod domain, and a microtubule-binding domain (MTBD). There are at least three major forms of BPAG1: BPAG1-e (302 kD), BPAG1-a (615 kD), and BPAG1-b (834 kD). BPAG1-e has been described previously and consists of the plakin domain, the CC rod domain, and the first IFBD. It is the primary epidermal BPAG1 isoform, and its absence that is the likely cause of skin fragility in mutant mice. BPAG1-a is the major isoform in the nervous system and a homologue of the microtubule actin cross-linking factor, MACF. BPAG1-a is composed of the ABD, the plakin domain, the SR-containing rod domain, and the MTBD. The absence of BPAG1-a is the likely cause of sensory neurodegeneration in mutant mice. BPAG1-b is highly expressed in muscles, and has extra exons encoding a second IFBD between the plakin and SR-containing rod domains of BPAG1-a.

Show MeSH
Related in: MedlinePlus