Figure 1.
Recruitment of Xbp1 mRNA to the ER membrane in mammalian cells.
XBP1unspliced originates translational pausing through its C-terminal region. The hydrophobic regions (HR1 and HR2) in XBP1unspliced target the XBP1 unspliced mRNA/ribosome/nascent chain complex to the ER membrane, giving the opportunity for Ire1 to splice Xbp1 mRNA. Adapted from [31].
Figure 2.
Conservation of HR2 and C-terminal region of Xbp1unpsliced.
(A) Kyte and Doolittle hydrophobicity plot of Drosophila Xbp1unspliced, indicating the existence of two hydrophobic regions, HR1 and HR2. The horizontal red line indicates a score of 1.8. (B) Local amino acid sequence alignment of HR2 using several species. Amino acids that are fully, strongly or weakly conserved are indicated. The hydrophobicity of each amino acid is indicated by a color code. (C) Local amino acid sequence alignment of the C-terminal translational pausing region. Amino acids that are fully, strongly or weakly conserved are indicated.
Figure 3.
Excision101 originates a truncated Xbp1 mRNA that is spliced by Ire1.
(A) Schematic representation of genomic region around Xbp1, with the localization of Excision101 deletion and the PCR primers Fwd1, Rev1, Rev2 and Rev3. In Excision 101, the breakpoint in P{SUPor-P}CG9418KG05183 is 5′GAA TTA CCT TGT AGT TGA TAT TTG AGA T (following the leading strand of Xbp1). (B) Schematic representation of the Xbp1unspliced and Xbp1spliced proteins in “wild-type” and in Excision101. PstI has a cleavage site in the intron spliced by Ire1. HR1: hydrophobic region 1. HR2: hydrophobic region 2. (C) Quantitative RT-PCR for total Xbp1 mRNA levels in larvae homozygous for Excision101or heterozygous Excision101/CyO-GFP, using the primers for Xbp1, Fwd1 and Rev3. Control Excision101/Cyo is set as 1, with the homozygous Excision101 Xbp1 mRNA levels indicated as a mean +/− standard deviation (a.u. - arbitrary units). (D) Agarose gel electrophoresis of RT-PCR products specific for Excision101 or CyO control chromosome after digestion with PstI restriction enzyme. PCR product specific for the Xbp1unspliced form is cleaved by PstI, while Xbp1spliced form is resistant to PstI digestion (because the PstI is in the intron that is spliced by Ire1). A positive control using genomic DNA is fully digested by PstI.
Figure 4.
cDNA sequencing demonstrates splicing in Excision101 specific mRNA.
Sequencing of single colonies obtained after cloning of Excision101 specific cDNA. The absence of the intron in colonies 1–4 demonstrates that Excision101 mRNA is spliced by Ire1. Percentage of colonies containing Xbp1spliced form in Excision101 or CyO control chromosome. The total number of colonies sequenced was 38 for Excision101 and 37 for the CyO control.
Figure 5.
Excision101 homozygous larvae have impaired UPR activation.
(A) Picture of 3-day old Excision101 homozygous larva and heterozygous Excision101/CyO-GFP sibling control. Excision101 homozygous larva arrest development during first instar stage. (B) Quantitative RT-PCR for BiP and Pdi mRNA levels in Excision101 homozygous or control heterozygous Excision101/CyO-GFP larvae treated with/without tunicamycin food. Induction of BiP and Pdi upon tunicamycin treatment is impaired in Excision101 homozygous larva. mRNA levels are indicated as a mean +/− standard deviation (a.u. - arbitrary units).