Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells
Figure 6
U2AF65/N-PU and hnRNP H/G2-G3 interplay in the N50 exon recognition.
(A) hnRNP H-RNA recognition is a prerequisite for U2AF65-RNA binding. The wild type RNA was incubated with whole (lane 2), hnRNP H-depleted (lane 3), and unrelated antigen-depleted H69 NEx (lane 4) or beads alone (lane 5). Then CLIP assays were carried out using U2AF65 antibodies and the recovered bound RNA was monitored by RT-PCR and quantified densitometrically. No RT (– RT) control PCR appears in lane 1. In the absence of hnRNP H, 60% less RNA was recovered with the U2AF65 antibodies, and no difference was observed in the absence of an unrelated antigen. (B) Kinetics of U2AF65- and hnRNP H-RNA binding. H69 NEx were incubated with wild type or mutant (G2-G3∼N-PU-less) RNA probes during the indicated times at room temperature. Then, CLIP assays were carried out with U2AF65 or hnRNP H antibodies and the recovered RNAs were amplified by RT-PCR (top panel, electrophoresis of the amplified products) and quantified densitometrically (plot at the bottom). At each time point, IntDen values obtained from the CLIP with the mutant RNA were subtracted from those obtained with wild type RNA in order to discriminate indirect protein-RNA interactions (e.g. mediated by additional factors) and unspecific binding to G-like and PU-like motifs. Significant amounts of hnRNP H-bound RNA are recovered as early as 4 min plummeting at 10 min, coinciding with the onset of U2AF65-bound RNA recovery. (C) TIA-1/TIAR is able to bind to multiple poly-U tracts. Different probes (wild type G2-G3∼N-PU, G2-G3∼mutant N-PU, and 3'ss control) were used in CLIP experiments as in B. Immunoprecipitations were carried out with antibodies against TIA-1/TIAR and U2AF65, and the recovered RNAs were amplified by RT-PCR and quantified. IntDen % represents the expression change of the indicated protein or mRNA compared to their respective control treatment. ND, not detected.