Specificity and determinants of Sam68 RNA binding - Implications for the biological function of K homology domains

Sam68, a specific target of the Src tyrosine kinase in mitosis, possesses features common to RNA-binding proteins, including a K homology (KH) domain, To elucidate its biological function, we first set out to identify RNA species that bound to Sam68 with high affinity using in vitro selection, From a degenerate 40-mer pool, 15 RNA sequences were selected that bound to Sam68 with K-d values of 12-140 nM. The highest affinity RNA sequences (K-d similar to 12-40 nM) contained a UAAA motif; mutation to UACA abolished binding to Sam68, Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding, The KH domain alone did not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding, Deletion of the KH domain or mutation of RR domain residues analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-l abolished G8-5 binding, Our results establish that a RB domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development.