Conserved and diverged mechanisms for RNA target-site recognition by the multi KH-domain Bicaudal C protein

Abstract

ABSTRACTThe Bicaudal-C (Bicc1) multi-KH domain RNA binding protein controls cell fates by binding and repressing the translation of specific target mRNAs. To provide new insights into Bicc1-RNA binding we used quantitative assays to analyze interactions between Xenopus Bicc1 protein and the 32-nt Bicc1 RNA target site from XlCripto-1 mRNA. High-affinity binding by XlBicc1(Kd=34nM) required both a 5’ leader sequence and a 3’loop sequence. Bicc1 proteins from humans (HsBicc1), zebrafish (DrBicc1) and fruit flies (DmBicc1) also bound the XlCripto-1 RNA with high-affinity via an interaction requiring the 5’ leader sequence, providing evidence for a conserved mechanism of RNA recognition by Bicc1. However, in contrast to XlBicc1, each of the other species’ Bicc1s showed a significantly relaxed requirement for the 3’ loop sequence. Analyses of mutant forms of XlBicc1, revealed that the KH1 domain promoted Bicc1’s requirement for the loop sequence via a GXXG-independent mechanism. In addition, a fusion protein comprised of the XlKH1 and the DmBicc1-KH2-KH3 subdomains was sufficient to establish high-affinity, loop-sensitive Bicc1-binding to XlCripto-1 RNA. These data support a model wherein the KH2 domain interacts with the leader sequence via a GXXG-mediated KH-RNA interface, defining the conserved core of the metazoan Bicc1-RNA interaction, while the KH1 domain is required for the formation of a more flexible, secondary protein-RNA interface that allows recognition of the 3’ loop sequence. This secondary interface differs between species and possibly between target mRNAs, establishing the potential for a range of translational repression activities for Bicc1 target mRNAs.