Figure 2: TDP-43 is essential for IL-6 pre-mRNA processing and stability.

(a,d) BMDMs or BMDCs were transfected with scrambled or TDP-43-specific siRNA. After treatment with LPS (80 ng ml⁻¹) (a) or CpG-DNA (1 μM) (d) for 6 h, ELISA assay was carried out. *P<0.01 (Student’s t-test). (b,e) RT–PCR for the exon–exon junction of the indicated cytokines was performed from LPS- or CpG-DNA-stimulated BMDMs expressing scrambled or TDP-43 siRNA. RT–PCR with the indicated primers (P1, P4, P6 or P7) for IL-6, TNF-α, TDP-43 and GAPDH yielded products of 167, 340, 405 and 376 bp, respectively, as predicted. P (No.) refers to the primer number for the indicated genes (Supplementary Fig. 2c and Supplementary Table 1). Arrowheads and arrows indicate mRNA and pre-mRNA, respectively. Quantification of the band intensity of mRNA and pre-mRNA of IL-6 or TNF-α (right graphs). *P<0.005 (Student’s t-test). (c) qRT–PCR using primer pairs (P2, P4 or P6) confirmed statistically significant reduction in IL-6 mRNA levels in TDP-43-depleted primary macrophages. All qRT–PCR data were normalized to GAPDH mRNA expression. *P<0.005 and **P<0.001 (Student’s t-test). (f) TDP-43 binds IL-6 pre-mRNA or mRNA, but not TNF-α pre-mRNA or mRNA. RAW macrophages expressing HA-TDP-43 were exposed to LPS (80 ng ml⁻¹) for 6 h. Lysates were immunoprecipitated with either anti-HA or anti-isotype antibody, and then subjected to RIP assay using the indicated primer pairs. RT–PCR with primer pairs (P1, P4 or P5) for IL-6 pre-mRNA/mRNA, TNF-α mRNA or TNF-α pre-mRNA, respectively, yielded 337/167, 340 or 448-bp products, as predicted for the RIP assay. Arrow and arrowheads indicate direct TDP-43 interaction with IL-6 pre-mRNA and mRNA. (g) The effect of TDP-43 depletion on pre-mRNA stability in LPS-stimulated macrophages was assessed by RT–PCR analysis of IL-6 pre-mRNA levels using primer pair P1. RNA was isolated at different time points after Act.D (5 μg ml⁻¹) treatment. Densitometric analysis of the bands obtained in control or TDP-43-depleted cells. (h) qRT–PCR revealed the presence of unstable IL-6 pre-mRNA in TDP-43-depleted RAW macrophages. *P<0.01 (Student’s t-test). Data are representative of more than three independent experiments (mean±s.d. in a, b, d and g; mean±s.e.m. in c and h).