Figure 6. Construction of mutant alleles to express various forms of XBP1.
(A) Schematic representation of the XBP1 gene as well as positions of the IRE1-mediated splice sites and three deletion mutants (Δ15, Δ26, Δ4). (B) Schematic representation of IRE1-mediated, unconventional splicing of XBP1 mRNA which removes the 26 b in exon 4 to produced pXBP1(S) as well as spliceosome-mediated alternative splicing of XBP1 mRNA which removes the entire exon 4 of 167 b to produce pXBP1(AS). pXBP1(U) is produced from unspliced XBP1 mRNA. (C) Schematic representation of pXBP1(U) (unspliced form), pXBP1(S) (spliced form) and pXBP1(AS) (alternatively spliced form). AD denotes transcriptional activation domain. (D) Sequences of a part of exon 4 in which three deletions were introduced by the TALEN method (Δ4 and Δ26) or CRISPR-Cas9 method (Δ15). The position of the XBP1 intron is also shown. (E) CentroidFold-predicted secondary structures of XBP1 mRNA around the IRE1-mediated splice sites (shown by two red arrows in a) produced by three deletion mutants (Δ15, Δ26, and Δ4) in comparison with those of unspliced and spliced XBP1 mRNA. The Δ15 and Δ26 deletion mutants produce constitutively expressed pXBP1(UC) and pXBP1(SC), respectively, which are not modified further after ER stress, as summarized in (F). The Δ4 deletion mutant produces pXBP1(Δ4) containing foreign 15 aa depicted as the grey box after aa165, which is inactive due to the absence of AD, but produces active pXBP1(AS) if spliceosome-mediated alternative splicing occurs, as summarized in (F). (F) Effects of ER stress and alternative splicing on expression of various forms of XBP1.