Binding of DAZ and DAZL1 to RNA Molecules
The abilities of DAZ and DAZL1 to bind RNA molecules were studied using an assay in which in vitro-synthe-sized 35S-labeled proteins were incubated with RNA homopolymers immobilized on agarose beads, and the bound proteins were analyzed by SDS-PAGE. As shown in Figure 1a, human DAZ and DAZL1, as well as mouse DAZL1, bound preferentially to poly U and poly G, similar to the Xenopus DAZL1 homologue, Xdazl, and FMRP. On the other hand, the protein product of an anonymous human cDNA clone KlAA0058 and the firefly lucif-erase, which contain no RNA binding motifs, failed to bind to any of the RNA homopolymers (data not shown). In the presence of increasing amounts of salt, the binding of DAZ and DAZL1 to poly U persisted, whereas the binding to poly G diminished, suggesting that DAZ and DAZL1 bind more strongly to poly U (Fig. 1b). These results verified the RNA-binding properties of DAZ and DAZL1. However, the biological significance of the preferential binding observed in the in vitro assays has yet to be demonstrated.
To delineate the regions required for RNA binding, a series of constructs with deletions in the RRM domain, the DAZ repeat region, or the C-terminal portion were generated (Fig. 2). These constructs were transcribed and translated in vitro, and the abilities of the resultant truncated proteins to bind poly U and poly G in the presence of 0.25 M NaCl were studied (Fig. 2). For DAZL1, deletion of RRM abolished its RNA-binding ability, whereas deletion of the single DAZ repeat affected the binding to poly G but not poly U, and that of the C-terminal portion showed little effect. It appears, therefore, that DAZL1 binds to RNA mainly through the RRM domain. The results for DAZ are less clear-cut. Deletion of RRM in either DAZ-R1 or DAZ-R2 diminished, but did not abolish, the RNA-binding ability. Similarly, deletion of seven of the nine DAZ repeats (in DAZ-Z1) or the C-terminal portion including the entire DAZ repeat region (in Z2) diminished but did not abolish the binding. The results suggest that the binding of DAZ to RNA does not depend solely on the RRM domain. To test whether the RRM domain and the C-terminal portion of DAZ can bind RNA homopolymers independently, fusion proteins were generated in which either the RRM domain or the C-terminal portion of DAZ was fused to the thioredoxin protein. Both fusion proteins bound to the RNA polymers, though at different strengths.
FIG. 1. In vitro binding of DAZ and DAZL1 to RNA homopolymers. a) Equal amounts of S35-labeled proteins were incubated with equal bed volumes of RNA homopolymers immobilized on agarose beads in the presence of 0.1 M NaCl. Bound proteins were analyzed by SDS-PAGE followed by autoradiography. The lanes are A, poly A; U, poly U; C, poly C; and G, poly G. b) The binding assay was carried out at the indicated salt concentration (Molar). The T lane contains an amount equivalent to 50% of the material used in the binding assay. DAZL, DAZL1.
FIG. 2. Mapping regions within DAZL1 and DAZ that are required for RNA binding. The structures of DAZL1 and DAZ and the regions retained in the truncated proteins are shown, with the RNA recognition motif (RRM) and the DAZ repeat region indicated. See Materials and Methods for the amino acid residues deleted in the proteins. Binding of proteins to poly U (U) and poly G (G) in the presence of 0.25 M NaCl are shown to the right. The T lane contains an amount equivalent to 50% of the material used in the binding reaction.