Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1′ and R8′, however, remain unclear. Here, we report how the repeats contribute to overall RNA binding. We first prepared three mutants in which R1′ and/or R8′ were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1′ contributed more than R8′, unlike Drosophila PUM. We next investigated which amino acid residues of R1′ or R8′ were responsible for RNA binding. With detailed analysis of the protein tertiary structure, we found a hydrophobic core in each of the repeats. We therefore mutated all hydrophobic amino residues in each core to alanine. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1′ had a significant influence. In the present study, we demonstrated that the flanking R1′ and R8′ repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1′-R1 interactions may stabilize the whole hPUM structure.
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