Regulation of messenger RNA is crucial in many contexts, including development, memory and cell growth. The 3' untranslated region is a rich repository of regulatory elements that bind proteins and microRNAs. Here we focus on PUF proteins, an important family of mRNA regulatory proteins crucial in stem-cell proliferation, pattern formation and synaptic plasticity. We show that two Caenorhabditis elegans PUF proteins, FBF and PUF-8, differ in RNA-binding specificity. FBF requires the presence of a single 'extra' nucleotide in the middle of an eight-nucleotide site, whereas PUF-8 requires its absence. A discrete protein segment is responsible for the difference. We propose that a structural distortion in the central region of FBF imposes the requirement for the additional nucleotide and that this mode of PUF specificity may be common. We suggest that new specificities can be designed and selected using the PUF scaffold.