Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional -helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nu-cleotides for GLD-1 (Caenorhabditis elegans). To un-derstand the structural basis of this probable differ-ence in specificity, we determined the solution struc-ture of GLD-1 KH-QUA2 with the complete consen-sus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a dif-ferent conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis show-ing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demon-strate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and con-tributes more generally to the modulation of RNA-binding affinity and specificity of KH domain con-taining proteins