FIGURE 6.

CUGBP1 and hnRNP H2 depletion result in a dependence on the poly(A) tail. CUGBP1 (A) and hnRNP H (B) were immunodepleted from rabbit reticulocyte lysate as described under ”Experimental Procedures.“ The depletion of these proteins was confirmed by immunoblotting (right) using antibodies against CUGBP1 (A) and hnRNP H (B). GAPDH served as a control for equal protein loading. The graphs on the left show the IRES activities of the bicistronic mRNAs containing a 30-nucleotide poly(A) tail (white bars) or lacking a poly(A) tail (dark bars) as measured in control rabbit reticulocyte, CUBBP1 (A)- or hnRNP H (B)-depleted rabbit reticulocyte lysate, or immunodepleted lysate supplemented with recombinant CUGBP1 (A) or hnRNP H2 (B). The relative ratio of Fluc/Rluc for each bicistronic mRNA containing a poly(A) tail was given a value of 1.0. The data represent the average of three independent experiments ± S.E. C, the 3′-UTR of the bicistronic mRNA was truncated by removal of nucleotides from the 3′-end, and the IRES activity of these truncation mutants was measured in rabbit reticulocyte lysate. The white bars represent the IRES activity of the truncated bicistronic mRNA containing a 300nucleotide poly(A) tail, and the shaded bars represent the IRES activity of the truncated bicistronic mRNA lacking a poly(A) tail. The relative ratio for each truncated bicistronic mRNA containing a poly(A) tail was given a value of 1.0. The data represent the average of three independent experiments ± S.E.