Abstract
Bruno protein binds to multiple sites - BREs - in the oskar mRNA 3' UTR, thereby controlling oskar mRNA translation. Bruno also binds and regulates other mRNAs, although the binding sites have not yet been defined. Bruno has three RRM type RNA binding motifs, two near the amino terminus and an extended RRM at the C terminus. Two domains of Bruno, the first two RRMs (RRM1+2), and the extended RRM (RRM3+) - can each bind with specificity to the oskar mRNA regulatory regions; these and Bruno were used for in vitro selections. Anti-RRM3+ aptamers include long, highly constrained motifs, including one corresponding to the previously identified BRE. Anti-RRM1+2 aptamers lack constrained motifs, but are biased towards classes of short and variable sequences. Bruno itself selects for several motifs, including some of those bound by RRM3+. We propose that the different RNA binding domains allow for combinatorial binding, with extended Bruno binding sites assembled from sequences bound by the individual domains. Examples of such sites were identified in known targets of Bruno, and shown to confer Bruno-dependent translational repression in vivo. Other proteins with multiple RRMs may employ combinatorial binding to achieve high levels of specificity and affinity.
