Fig. 3: GCLC is a target of the RIDD activity of IRE1α.
From: IRE1α determines ferroptosis sensitivity through regulation of glutathione synthesis
A Cellular reduced glutathione (GSH) levels in control (non-targeting) and IRE1α-null MDA-MB-231 cells overexpressing luciferase (Luc), wild-type (WT) or the K907A mutant IRE1α after 2.5 μM erastin treatment (12 h). B qRT-PCR analysis of GCLC mRNA expression in MDA-MB-231 cells with different IRE1α expression after DMSO or 10 μM erastin treatment (8 h). β-actin: normalization control. C Western blotting analysis of ferroptosis-related proteins in MDA-MB-231 cells with variable IRE1α expression ± erastin (10 μM, 6 hours). GFP: overexpression control. β-actin: loading control. D Left: schematic view of human GCLC transcript with coding sequence (CDS, green), exon boundaries (yellow), and fragments for in vitro transcription (light brown). The sequence flanking the identified IRE1α cleavage site is also shown with the GC cleavage site (red), loop sequence (green) and stem sequence (light blue). The pairing bases in the stem part are marked with black underlines and unpaired bases are marked with black loops. Right: the predicted secondary structure of the mRNA sequence flanking the GCLC IRE1α cleavage site (red). The black triangle marks the cleavage between G and C. E In vitro RNA cleavage assay using full length (wild-type and cleavage site mutated) and two large fragments of human GCLC mRNA (#1 and #2 as shown in D), incubated in the presence or absence of recombinant cytosolic portion of IRE1α (rIRE1α) ± IRE1α inhibitor 4μ8c (30 μM). Nucleotide base markers are shown on the left side. F. In vitro RNA cleavage assay using three smaller fragments of human GCLC mRNA (#3, #4 and #5 as shown in D). G In vitro RNA cleavage assay using wild-type and cleavage site-mutated (GC to CC) fragment #4. H In vitro RNA cleavage assay using wild-type and cleavage site-mutated (GC to CC) 29-base RNA probes within fragment #4. In (A) and (B), data are presented as mean ± s.d., n = 4 independent repeats. Unpaired, two-tailed Student’s t tests were performed to calculate the P values for all the statistical analyses. N.S. not significant. Source data are provided as a Source Data file.
