The V5 epitope tag coding sequence caused mis-splicing of DUX4 mRNA.

<p>A: <i>Top</i> shows the exon-intron structure of the DUX4 gene arising from the distal D4Z4 unit, and flanking pLAM region. Exons and introns are indicated but not to scale. The asterisk (*) represents the normal DUX4 stop codon. The final 11 nucleotides of exon 1 are untranslated, as are those in exons 2 and 3 (the latter is part of the pLAM sequence). In an FSHD permissive genotype, the DUX4 polyA signal is located in exon 3. <i>Bottom</i> shows the same locus but with the V5 coding sequences inserted. A new stop codon was placed downstream of the V5 tag, followed by 40 nucleotides of linker sequences (N40). The lower case “g” indicated by an arrow and red text, is the aberrant splice site created by the DUX4-V5 fusion coding sequence. B: AAV.D4Z4.V5 vectors express DUX4.V5 mRNA. A previously reported nested PCR/3’ RACE strategy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118813#pone.0118813.ref024" target="_blank">24</a>] was used to amplify 3’ end of the DUX4 transcript from RNAs harvested from AAV.D4Z4.V5-injected C57BL/6 TA muscles. A ~450 bp band was amplified from muscles that received AAV.D4Z4.V5 but not AAV.GFP controls. The size of this band corresponded to the expected product of the reverse transcribed and PCR-amplified DUX4 3’ end if introns 1 and 2 were spliced out. C: Sequence chromatogram of cloned 3’ RACE products identifying the mis-splicing event. The arrow points out the same g nucleotide indicated in panel A. D: The V5 epitope, N40 linker sequences, and intron 1 are spliced out of the D4Z4.V5 transcript. E: This mis-splicing event created a new missense DUX4 protein containing an additional 55 amino acids (DUX4-55aa), until an in-frame stop codon occurred in exon 3.