An Abundant Evolutionarily Conserved CSB-PiggyBac Fusion Protein Expressed in Cockayne Syndrome

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.

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

“…Because of the "absolute" nature of Ct shift resulting signal, the method also allows to calculate indirectly determined transcript levels by subtraction or addition of different target RQs, in this case differentiating between promoter activities and alternative splicing. Using this approach, we could show remarkable variability of the cryptic promoter usage among cell types ( Figure 4C), and the comparable expression of the "host" (CSB) mRNA and the CSB-PGBD3 fusion product ( Figure 3C), further supporting the importance of the domesticated PGBD3 expression, despite its as yet undetermined function (Newman et al, 2008). It is clearly seen that higher variance is observed among cell types for the PGBD3 splice variants than normal CSB splice variants; moreover, we can suspect important function of the PGBD3 splice product from the cryptic promoter in the MSCL-2 fibroblast cell line, as its expression is significantly higher than in the other cell lines ( Figure 4B).…”

“…It is clearly seen that higher variance is observed among cell types for the PGBD3 splice variants than normal CSB splice variants; moreover, we can suspect important function of the PGBD3 splice product from the cryptic promoter in the MSCL-2 fibroblast cell line, as its expression is significantly higher than in the other cell lines ( Figure 4B). In fact, an autonomous PGBD3 protein product corresponding to this splice variant has been already detected by western blots (Newman et al, 2008), underlining its potential function. Although we also detected a significant level of the "normal" CSB splice product from this cryptic promoter, it is currently unknown if it is translated.…”

“…The Ct shift method offers a good solution for monitoring the levels of these gene products, and this could be applied for splice variants of certain domesticated transposase genes whose cellular functions have only been partially revealed (Sinzelle et al, 2009) or have not yet been elucidated (Alzohairy et al, 2013;Vogt et al, 2013). Some members of the PiggyBac transposase family express themselves by a gene trapping strategy, and in the absence of an own promoter they can only be transcribed as an alternative mRNA fusion product from the host (Newman et al, 2008). PGBD3, one of the PiggyBac derived transposons in the human genome is abundantly expressed in such a fusion form with Cockayne syndrome B (CSB) protein (http://biogps.org/).…”

“…CSB protein has functions in transcription-coupled nucleotide excision repair (Sarker et al, 2005;Laine and Egly, 2006) and chromatin remodeling (Newman et al, 2006), and mutations of the CSB gene are responsible for 70% of CS disease (Laugel et al, 2010). PGBD3 has been conserved in the 5th intron of CSB gene in Primates, and alternative splicing produces a protein fusion product with the N-terminal part of CSB protein (Newman et al, 2008). In addition, using a semiquantitative method, the authors showed that a cryptic transcript is also expressed starting at the end of exon 5 (Newman et al, 2008).…”

“…PGBD3 has been conserved in the 5th intron of CSB gene in Primates, and alternative splicing produces a protein fusion product with the N-terminal part of CSB protein (Newman et al, 2008). In addition, using a semiquantitative method, the authors showed that a cryptic transcript is also expressed starting at the end of exon 5 (Newman et al, 2008). The role of the PGBD3 transposon as part of these transcripts is not known but it is probably beneficial for normal cell physiology.…”