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.