(A) Crystal structure of an FBF-2/LST-1/RNA ternary complex. FBF-2 is shown as a ribbon diagram with cylindrical helices. PUM repeats are colored alternately red and blue. RNA recognition side chains from each PUM repeat are shown with dotted lines indicating interactions with the RNA bases. LST-1 (green) and the RNA (beige) are shown as stick representations colored by atom type (red, oxygen; blue, nitrogen; orange, phosphorus). (B) LST-1 contacts FBF-2 at conserved interaction hotspots. Zoomed-in view of interaction between FBF-2 and LST-1. Three interaction hotspots are labeled, and LST-1 L83 and L76 at hotspots 1 and 3, respectively, are shown with space-filling atoms. LST-1 K80 and FBF-2 Q448 at hotspot 2 are shown as stick models. Interactions between LST-1 and FBF-2 are indicated by dotted lines. Electron density for the LST-1 peptide is shown in Figure 2—figure supplement 1. (C) Conservation of LST-1 interacting residues in CPB-1 and GLD-3. Amino acid sequence alignment of the LST-1 interacting peptide and conserved sequences in CPB-1 and GLD-3. Residues at the interaction hotspots in (B) are highlighted and conserved residues are in boldface. (D) LST-1 L83 and Y85 at interaction hotspot 1 are essential for tight binding to FBF-2. Yeast 2-hybrid analyses were conducted with LST-1 residues 55–105 fused to the LexA DNA-binding domain (D.B.D.) and the PUM domain of FBF-2 fused to the GAL4 activation domain (A.D.). Mutants in LST-1 that interfered with FBF-2 interaction are colored green and those that were competent for interaction are colored gray. (E) FBF-2 Q448G at hotspot 2 has a minor effect on interaction with LST-1. FBF-2 variants that interfered with LST-1 interaction are colored red and those that were competent for interaction are colored gray. Binding activity is shown as units of β-gal activity normalized to cell count. Error bars indicate the standard deviation of three biological replicate measurements.