PMC

hnRNP A18 binds to TRX 3′-UTR in solution. (A) Schematic representation of the different probes used. FL; full-length, nt; nucleotides. (B) RNA band shift was performed as described in the text. Increasing amounts (0.12 to 4 µg) of GST–hnRNP A18 was incubated with the indicated labeled TRX probe and run on a 1% agarose gel. (C) hnRNP A18 RBD and RGG domain are required for maximal RNA binding. Northwestern analysis was performed with increasing amounts (0.5 to 2 µg) of either full-length (FL) GST–hnRNP A18 (lanes 8–10), GST–hnRNP A18 RGG domain (RGG; lanes 2–4), GST–hnRNP A18 RBD (lanes 5–7) or GST alone (lane 1) and labeled TRX 3′-UTR as described in the text.

Phosphorylation of hnRNP A18. (A) In vitro phosphorylation with recombinant hnRNP A18 and recombinant CK2 (lane 10), GSK3α (lane 1), GSK3β (lane 8) and FLAG immunoprecipitated SMG1^ATX (lane 9), ATM (lane 4), ATR (lane 3) as described in the text. The positions of phosphorylated hnRNP A18 and autophosphorylated GSK3 and CK2 are indicated. (B) Northwestern analysis was performed with increasing amounts (0.25 to 0.5 µg) of recombinant hnRNP A18 phosphorylated in vitro with cold ATP and the indicated kinases (SMG1^ATX; lanes 5 and 6, GSK3β; lanes 3–4, CK2; lanes 1–2). The blot was reacted with labeled TRX 3′-UTR.

hnRNP A18 protein levels correlate with TRX protein levels. (A) Western blots analysis performed on RKO protein extracts (50 µg) exposed or not to the indicated dose of UV radiation. The ‘Fold’ induction was measured by densitometry analysis and normalized to Actin. (B) Western blot analysis of RKO cells stably transfected with an hnRNP A18 antisense vector (A18-AS) or an empty vector (vector) and exposed to the indicated dose of UV radiation. (C) Western blots analysis of RKO cells transiently transfected (+) or not (−) with hnRNP A18 siRNA (A18-Si) or scrambled RNA (Cont-Si) and exposed to the indicated dose of UV radiation. Actin is used as a loading control. TRX: thioredoxin. Densitometry analysis is expressed as ‘Fold’ induction normalized to Actin and then to cells transfected with hnRNP A18 siRNA.

hnRNP A18 interacts with TRX mRNA in the cells. (A) Polysomes were extracted from RKO cells exposed to 20 J m⁻² UV radiation. IP with either anti IgG or anti-GFP to pull down GFP-A18 hnRNP (A18) was performed in the presence of 20 mM EDTA to disrupt the polysomes but preserve the association of RBP with target mRNAs. IP was followed by RT–PCR to detect endogenous TRX. As a negative control, VEGF, GADD45 and GAPDH were amplified from the same fractions. To facilitate detection of GAPDH, three times as much material as the TRX products was loaded. (B) Same as (A) except that anti hnRNP A18 was used to pull down mRNAs bound to endogenous hnRNP A18.