Intronic Binding Sites for hnRNP A/B and hnRNP F/H Proteins Stimulate Pre-mRNA Splicing
Figure 2
The hnRNP A1 Protein Stimulates Large-Intron Splicing
(A) Sequestering hnRNP A/B proteins affects large-intron splicing. The large-intron substrates 7-AdB lacking ABS (−.−) or containing ABS (a.a) were co-incubated with the small-intron 7B-Ad pre-mRNA (80 pM for the 7-AdB substrates and 16 pM for the control 7B-Ad pre-mRNA) in a HeLa extract for 90 min in the presence of increasing amounts of the telomeric oligonucleotide TS10 (0, 80, 160, 320, and 640 nM, respectively).
(B) Splicing mixtures were incubated with increasing amounts of recombinant GST-A1 protein (0, 0.8, 1.6, and 3.2 μM). The 7-AdB pre-mRNA carrying inverted repeats (→.←) was also used.
(C) Splicing mixtures containing the 7-AdB and the small-intron 7B-Ad were supplemented with His-A1 and GST-A1 (0.5 μM each).
(D) The histogram depicts a compilation of three independent experiments performed with the indicated concentrations of recombinant proteins. In each case, individual values obtained for the splicing of 7-AdA (−.−) or 7-AdA (a.a) pre-mRNAs were normalized with the splicing efficiency of the small intron 7B-Ad pre-mRNA. Error bars indicate standard deviations.