The repertoire of binding specificities for two repeats in the RNA-binding domain of Drosophila Pumilio

Pumilio consists of eight homologous repeats that act in a modular fashion to recognize Nanos response elements (NREs) in targeted mRNAs, each repeat interacting with a single base of the NRE. Most of the sequence specificity of binding is thought to be driven by interactions between residues 12 and 16 of each repeat with the edge of the appropriate RNA base. In this report, we investigate the repertoire of amino acids at positions 12 and 16 that are capable of mediating high-affinity binding for two of the PUF repeats, R6 and R5. We generate plasmid libraries in which the codons for residues 12 and 16 are randomized, transform these into a suitable yeast strain, and screen for variants that recognize an NRE in three-hybrid RNA-binding experiments. We find that the two repeats have very different capabilities. Relatively few amino acid combinations in R6 are functional, and these exhibit a limited array of binding specificities; in contrast, ∼24% of the edge-on amino acid combinations in R5 are functional, and these exhibit a variety of novel specificities. These results support the idea that R6 (in part) defines NRE target sites, while R5 selects among NREs to allow differential regulation in vivo. We also show that R5 binding modules generally cannot be functionally swapped into R6. Thus, although binding of the Pumilio PUF domain is modular, the R6 and R5 modules are not readily interchangeable, consistent with studies of other PUF domains.