Fig. 7: IRE1α suppresses Thrombospondin 1 expression through its RIDD activity.
a MS1 cells were pre-cultured at 5 mM glucose for 4 hours before stimulation with 16 mM glucose for 12 hours in the absence or presence of 10 μM 4μ8C. Quantitative RT-PCR analysis of Xbp1 mRNA splicing and Thbs1 mRNA abundance (n = 3 independent experiments). b HEK293T cells with IRE1α depletion (HEK293T-KO) were transiently transfected for 36 hours with vector control (-) or THBS1-Myc plasmid, or co-transfected with THBS1-Myc plus IRE1α or XBP1s-Flag plasmids. Cells transfected as indicated were also treated with 10 μM 4μ8C for 12 hours. Immunoblot analysis of IRE1α, XBP1s and TSP1-Myc protein. Shown also is quantification of TSP1-Myc protein level after normalization to β-Actin control (n = 3 independent experiments). c, d HEK293T-KO cells were transiently transfected for 36 hours with vector control, IRE1α-WT or its RNase-deficient K907A mutant plasmids. c Quantitative RT-PCR analysis of human XBP1s, BLOC1S1 and THBS1 mRNA levels (n = 3 independent experiments). d Immunoblot analysis of IRE1α, TSP1-Myc and XBP1 protein with α-Tubulin as a loading control. Shown also is quantification of TSP1-Myc protein levels (n = 3 independent experiments). e Consensus sequence (underlined) of the putative RIDD region in human and mouse TSP1 mRNAs within the stem-loop structure predicted by RNAstructure Version 6.2. f Agarose gel analysis of IRE1α-mediated cleavage of human THBS1 mRNA. In vitro transcription-derived mRNA fragments of THBS1 (nt 1-1770 and nt 1771-3510) were incubated for 1 hour with recombinant human IRE1α protein (1 μg) in the presence of DMSO (-) or 10 μM 4μ8C, followed by 3% agarose gel analysis. Human XBP1 mRNA was used as a positive control. Red arrows indicate the probable major RNA cleavage products. g Synthetic RNA substrates for wild-type THBS1WT (nt 2771-2808) or THBS1Mut with the indicated G-to-C mutation were incubated for 1 hour with human IRE1α protein (1 μg) pre-mixed for 1 hour with DMSO (-) or 10 μM 4μ8C, followed by 20% TBE-Urea PAGE gel analysis. The red arrow indicates the RNA cleavage product. Data are representative of 2 independent experiments in (f, g). Results are shown as mean ± SEM by unpaired two-tailed Student’s t-test (a–d).
