RNA binding specificity of Unr, a protein with five cold shock domains

The human unr gene encodes an 85 kDa protein which contains five cold shock domains (CSD), The capacity of Unr to interact in vitro with RNA and its i ntracellular localization suggest that Unr could be involved in some aspect of cytoplasmic mRNA metabolism. As a step towards identification of Unr mR NA targets, we investigated the RNA-binding specificity of Unr by an in vit ro selection approach (SELEX). Purine-rich sequences were selected by Unr, leading to the identification of two related consensus sequences characteri zed by a conserved core motif AAGUA/G or AACG downstream of a purine stretc h. These consensus sequences are 11-14 nt long and appear unstructured. RNA s containing a consensus sequence were bound specifically by Unr with an ap parent dissociation constant of 1 x 10(-8) M and both elements, the 5' puri ne stretch and the core motif, were shown to contribute to the high affinit y. When the N-terminal and C-terminal CSD were analyzed individually, they exhibited a lower affinity than Unr for winner sequences (5- and 100-fold, respectively) but with similar binding specificity, Two combinations of CSD s, CSD1-2-3 and CSD1(star)2-3-4-5 were sufficient to achieve the high affin ity of Unr, indicating some redundancy between the CSDs of Unr for RNA reco gnition, The SELEX-generated consensus motifs for Unr differ from the AACAU C motif selected by the Xenopus Y-box factor FRGY2, indicating that a diver sity of RNA sequences could be recognized by CSD-containing proteins.