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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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The Extent and Abundance of the CSB-PGBD3 Fusion Transcript Assayed by Quantitative Real-Time RT-PCR.(A) Location of six upstream primer sites, common to both the CSB and CSB-PGBD3 fusion transcripts; and three downstream primer sites specific to each transcript. (B) Most of the tested primer pairs generated clean PCR products of the predicted size; identity was verified by sequencing. The smaller products generated by primers A, D, E and F appear to be alternatively spliced transcripts lacking the 745 nt CSB exon 5; these transcripts are not predicted to encode a functional protein. The products of the 40-cycle real-time protocol were resolved by 1% agarose gel electrophoresis. K, Invitrogen 1 kb ladder; C, NEB 100 bp ladder; markers in bp.
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pgen-1000031-g002: The Extent and Abundance of the CSB-PGBD3 Fusion Transcript Assayed by Quantitative Real-Time RT-PCR.(A) Location of six upstream primer sites, common to both the CSB and CSB-PGBD3 fusion transcripts; and three downstream primer sites specific to each transcript. (B) Most of the tested primer pairs generated clean PCR products of the predicted size; identity was verified by sequencing. The smaller products generated by primers A, D, E and F appear to be alternatively spliced transcripts lacking the 745 nt CSB exon 5; these transcripts are not predicted to encode a functional protein. The products of the 40-cycle real-time protocol were resolved by 1% agarose gel electrophoresis. K, Invitrogen 1 kb ladder; C, NEB 100 bp ladder; markers in bp.

Mentions: We were able to detect the predicted CSB-PGBD3 fusion transcripts by quantitative, real-time RT-PCR (Q-RT-PCR) using HeLa mRNA as template, forward primers for the 3′ half of CSB exon 5 which is shared by the CSB and predicted fusion mRNAs, and reverse primers which are specific for either CSB exon 6 or the PGBD3 element ( 2A). The fusion products exhibited the expected size ( 2B) and sequence (data not shown), and were approximately 2-fold more abundant than the equivalent CSB products (Figure S1). Moreover, we readily detected fusion products using forward primers for CSB exons 2, 3 and 4, indicating that a significant fraction of the CSB-PGBD3 fusion transcripts initiate far upstream of the putative cryptic promoter, presumably at a natural CSB initiation site. These full-length CSB-PGBD3 fusion transcripts do not reflect template strand switching by reverse transcriptase or recombination during PCR [37] within exon 5, because alternatively spliced fusion transcripts lacking exon 5 were also observed (Figure 2), and the abundance of the fusion products was not diminished in control experiments where either one of the potentially recombining mRNAs was sequestered within a cDNA:mRNA hybrid by a preliminary reverse transcription step using an mRNA-specific primer (data not shown).


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)

The Extent and Abundance of the CSB-PGBD3 Fusion Transcript Assayed by Quantitative Real-Time RT-PCR.(A) Location of six upstream primer sites, common to both the CSB and CSB-PGBD3 fusion transcripts; and three downstream primer sites specific to each transcript. (B) Most of the tested primer pairs generated clean PCR products of the predicted size; identity was verified by sequencing. The smaller products generated by primers A, D, E and F appear to be alternatively spliced transcripts lacking the 745 nt CSB exon 5; these transcripts are not predicted to encode a functional protein. The products of the 40-cycle real-time protocol were resolved by 1% agarose gel electrophoresis. K, Invitrogen 1 kb ladder; C, NEB 100 bp ladder; markers in bp.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000031-g002: The Extent and Abundance of the CSB-PGBD3 Fusion Transcript Assayed by Quantitative Real-Time RT-PCR.(A) Location of six upstream primer sites, common to both the CSB and CSB-PGBD3 fusion transcripts; and three downstream primer sites specific to each transcript. (B) Most of the tested primer pairs generated clean PCR products of the predicted size; identity was verified by sequencing. The smaller products generated by primers A, D, E and F appear to be alternatively spliced transcripts lacking the 745 nt CSB exon 5; these transcripts are not predicted to encode a functional protein. The products of the 40-cycle real-time protocol were resolved by 1% agarose gel electrophoresis. K, Invitrogen 1 kb ladder; C, NEB 100 bp ladder; markers in bp.
Mentions: We were able to detect the predicted CSB-PGBD3 fusion transcripts by quantitative, real-time RT-PCR (Q-RT-PCR) using HeLa mRNA as template, forward primers for the 3′ half of CSB exon 5 which is shared by the CSB and predicted fusion mRNAs, and reverse primers which are specific for either CSB exon 6 or the PGBD3 element ( 2A). The fusion products exhibited the expected size ( 2B) and sequence (data not shown), and were approximately 2-fold more abundant than the equivalent CSB products (Figure S1). Moreover, we readily detected fusion products using forward primers for CSB exons 2, 3 and 4, indicating that a significant fraction of the CSB-PGBD3 fusion transcripts initiate far upstream of the putative cryptic promoter, presumably at a natural CSB initiation site. These full-length CSB-PGBD3 fusion transcripts do not reflect template strand switching by reverse transcriptase or recombination during PCR [37] within exon 5, because alternatively spliced fusion transcripts lacking exon 5 were also observed (Figure 2), and the abundance of the fusion products was not diminished in control experiments where either one of the potentially recombining mRNAs was sequestered within a cDNA:mRNA hybrid by a preliminary reverse transcription step using an mRNA-specific primer (data not shown).

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