High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking

Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins.The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA binding by STAR proteins will help facilitate the identification of novel regulatory targets in vivo.Gene expression is regulated at the post-transcriptional level as a means of ensuring the proper localization and timing of developmental processes in eukaryotic organisms [1]. In a diverse set of pathways, a central feature of this post-transcriptional control involves specific binding to target RNA sequences by proteins belonging to the Signal Transduction and Activation of RNA/GRP33, Sam68, GLD-1 (STAR/GSG) family. STAR protein-RNA interactions are important for translational silencing of genes necessary for germline fate in hermaphrodite C. elegans worms by the regulatory STAR protein GLD-1 [2,3], mRNA localization and subsequent development of central nervous system components in mice by Quaking (Qk-1) [4-6] and pre-mRNA binding by yeast BBP (Branchpoint sequence Binding Protein) and mammalian splicing factor 1 (SF-1) proteins as precursors for splicing into mature mRNA [7,8].Members of the STAR/GSG protein family share a high degree of sequence sim