Figure 3.
Uniquely conserved sequences in the RRM3 that are responsible for ELAV–ELAV interaction. (A) An amino-acid sequence alignment of RRMs from three Drosophila, and four human ELAV-family proteins; ELAV, Rbp9, Fne, HuR, HuB, HuC and HuD. Amino acids that are conserved among RRM3s but not at the same sites in RRM1 or 2 (RRM3-specific conservations) are indicated by magenta. Conserved amino acids among the RRM3s that are not specific to RRM3 are shown by blue. Darker color indicates identical amino acids to those of ELAV RRM3, and lighter color indicates conservative substitutions. Putative secondary structural elements are shown above. The seven underlined sequences were chosen for the mutational analysis. (B) The effect of mutations on the interaction. The hinge-RRM3 construct was mutated by replacing one of the seven underlined sequences with the corresponding sequence of RRM2. The interactions between the mutants and the wild-type hinge-RRM3 were tested by the two-hybrid assay. In the figure, the constructs above the ‘+’ or ‘−’ box are activation-domain fusions, and the eH3 ‘bait’ construct on the left is a DNA-binding-domain fusion. The loss of interaction activity of the mutants eH3mu3, 4 and 6 (columns 4, 5 and 7) suggests that the mutated amino acids are essential for the interaction. Introduction of the three sequences, 3, 4 and 6, to RRM2 (eH3mu8 construct, column 9) was sufficient to give the interaction activity to the hinge-RRM2. Photographs of representative testing and control plates are shown below.