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An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome.

Newman JC, Bailey AD, Fan HY, Pavelitz T, Weiner AM - PLoS Genet. (2008)

Bottom Line: The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase.The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya.Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3' terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.

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Related in: MedlinePlus

CSB-PGBD3 Fusion Protein Reacts with Anti-PGBD3 Antibody.Western blot of HT1080 extracts using CSB C-terminal and N-terminal antibodies, and a commercial antipeptide antibody to PGBD3. The PGBD3 antibody reacts with the same 140 kDa band as the N-terminal CSB antibody. CSB decreases relative to CSB-PGBD3 fusion protein as cells approach confluence; compare logarithmically growing HT1080 (Figure 3B, leftmost lane) with confluent cells (Figure 4, middle panel). The lower band on the PGBD3 panel is likely PGBD3 itself (predicted 68 kDa, presumably initiating at the cryptic promoter within CSB exon 5), while the middle 105 kDa band may reflect crossreaction with PGBD1 (predicted 93 kDa) which contains a region homologous to the peptide epitope. Markers in kDa.
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pgen-1000031-g004: CSB-PGBD3 Fusion Protein Reacts with Anti-PGBD3 Antibody.Western blot of HT1080 extracts using CSB C-terminal and N-terminal antibodies, and a commercial antipeptide antibody to PGBD3. The PGBD3 antibody reacts with the same 140 kDa band as the N-terminal CSB antibody. CSB decreases relative to CSB-PGBD3 fusion protein as cells approach confluence; compare logarithmically growing HT1080 (Figure 3B, leftmost lane) with confluent cells (Figure 4, middle panel). The lower band on the PGBD3 panel is likely PGBD3 itself (predicted 68 kDa, presumably initiating at the cryptic promoter within CSB exon 5), while the middle 105 kDa band may reflect crossreaction with PGBD1 (predicted 93 kDa) which contains a region homologous to the peptide epitope. Markers in kDa.

Mentions: (A) Western blot using a C-terminal antibody against CSB reveals the expected 170 kDa major band for full length CSB (filled arrowhead). CSB is weakly expressed in hTERT-immortalized WI38 cells, but not at all in hTERT-immortalized CS1AN cells (CSB-) until rescue with CSB cDNA (CSB-wt). (B) Western blot using an N-terminal antibody against CSB reveals both full length CSB (filled arrowhead) and, in all cell lines, a second major band of approximately 140 kDa (hollow arrowhead) that we identify in Figure 4 as a CSB-PGBD3 fusion protein. The fusion protein is more abundant than CSB in WI38/hTERT cells, and is strongly expressed in immortalized CS1AN cells before and after rescue. CS1AN is a compound heterozygote line; one CSB allele contains an early truncating mutation (K337STOP) but a second allele with a 100 nt deletion in exon 13 should permit expression of the fusion protein. CSB and the CSB-PGBD3 fusion protein both migrate more slowly than predicted based on molecular mass alone, presumably due to the N-terminal acidic domain (see text). Markers in kDa.


An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome.

Newman JC, Bailey AD, Fan HY, Pavelitz T, Weiner AM - PLoS Genet. (2008)

CSB-PGBD3 Fusion Protein Reacts with Anti-PGBD3 Antibody.Western blot of HT1080 extracts using CSB C-terminal and N-terminal antibodies, and a commercial antipeptide antibody to PGBD3. The PGBD3 antibody reacts with the same 140 kDa band as the N-terminal CSB antibody. CSB decreases relative to CSB-PGBD3 fusion protein as cells approach confluence; compare logarithmically growing HT1080 (Figure 3B, leftmost lane) with confluent cells (Figure 4, middle panel). The lower band on the PGBD3 panel is likely PGBD3 itself (predicted 68 kDa, presumably initiating at the cryptic promoter within CSB exon 5), while the middle 105 kDa band may reflect crossreaction with PGBD1 (predicted 93 kDa) which contains a region homologous to the peptide epitope. Markers in kDa.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2268245&req=5

pgen-1000031-g004: CSB-PGBD3 Fusion Protein Reacts with Anti-PGBD3 Antibody.Western blot of HT1080 extracts using CSB C-terminal and N-terminal antibodies, and a commercial antipeptide antibody to PGBD3. The PGBD3 antibody reacts with the same 140 kDa band as the N-terminal CSB antibody. CSB decreases relative to CSB-PGBD3 fusion protein as cells approach confluence; compare logarithmically growing HT1080 (Figure 3B, leftmost lane) with confluent cells (Figure 4, middle panel). The lower band on the PGBD3 panel is likely PGBD3 itself (predicted 68 kDa, presumably initiating at the cryptic promoter within CSB exon 5), while the middle 105 kDa band may reflect crossreaction with PGBD1 (predicted 93 kDa) which contains a region homologous to the peptide epitope. Markers in kDa.
Mentions: (A) Western blot using a C-terminal antibody against CSB reveals the expected 170 kDa major band for full length CSB (filled arrowhead). CSB is weakly expressed in hTERT-immortalized WI38 cells, but not at all in hTERT-immortalized CS1AN cells (CSB-) until rescue with CSB cDNA (CSB-wt). (B) Western blot using an N-terminal antibody against CSB reveals both full length CSB (filled arrowhead) and, in all cell lines, a second major band of approximately 140 kDa (hollow arrowhead) that we identify in Figure 4 as a CSB-PGBD3 fusion protein. The fusion protein is more abundant than CSB in WI38/hTERT cells, and is strongly expressed in immortalized CS1AN cells before and after rescue. CS1AN is a compound heterozygote line; one CSB allele contains an early truncating mutation (K337STOP) but a second allele with a 100 nt deletion in exon 13 should permit expression of the fusion protein. CSB and the CSB-PGBD3 fusion protein both migrate more slowly than predicted based on molecular mass alone, presumably due to the N-terminal acidic domain (see text). Markers in kDa.

Bottom Line: The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase.The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya.Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3' terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.

Show MeSH
Related in: MedlinePlus