Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Abstract

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

Fig. 1.

Crystal structure of FBF-2 in complex with gld-1 FBEa RNA. (A) Stereo-view of FBF-2 in complex with gld-1 FBEa RNA. Repeats are colored alternately red and blue. Side chains that interact with RNA are shown. The RNA is colored by atom type (gray, carbon; red, oxygen; blue, nitrogen; orange, phosphorus; yellow, sulfur). Bases 4–6 are shown with green carbon atoms. (B) Surface representation of FBF-2 in complex with gld-1 FBEa RNA. The figures were prepared with PyMol (44).

Fig. 2.

Recognition of conserved RNA regions by FBF-2. (A) Conservation of PUF protein recognition of the 5′-conserved UGU sequence. Superposition of repeats 6–8 in the structures of FBF-2 in complex with gld-1 FBEa RNA and PUM1 with NRE RNA. FBF-2 is depicted as in Fig. 1A. The NRE RNA and RNA-interacting side chains of PUM1 are colored yellow. Dashed lines indicate interacting atoms (gray, FBF; yellow, PUM1). Corresponding CA atoms in repeats 6–8 of FBF-2 and PUM1 were aligned (rmsd of 1.41 Å over 105 CA atoms). The RNA-interacting side chains of PUM1 are labeled in parentheses. (B) Conservation of PUF protein recognition at the 3′-end. Corresponding CA atoms in repeats 1–3 of FBF-2 and PUM1 were aligned (rmsd of 1.70 Å over 99 CA atoms).

Fig. 3.

Three classes of RNA conformation in FBF-2 target RNAs. Interaction of FBF-2 repeats 4–6 with gld-1 FBEa (A), fem-3 PME (B), and gld-1 FBEa G4A mutant (C) RNAs are shown. Dashed lines indicate interacting atoms. Water molecules that mediate interactions are shown as red spheres. (D) Superposition of the RNA-interacting side chains of FBF-2 repeats 4–6 and gld-1 FBEa (gray), fem-3 PME (tan), and gld-1 FBEa G4A mutant (pale cyan) RNAs (rmsd of 0.15 Å over 130 CA atoms between PME and FBEa and rmsd of 0.28 Å over 130 CA atoms between PME and FBEa G4A mutant).

Fig. 4.

Defining FBF RNA-binding specificity. (A) Superposition of the CA traces of FBF-2 (red) and PUM1 (yellow) aligning repeats 5–8. (B) Superposition of the CA traces of FBF-2 (red) and yeast Puf4p (green) aligning repeats 5–8. (C) Superposition of the CA traces of FBF-2 (red) and N-terminal (blue) and C-terminal (yellow) halves of PUM1. The separated halves of PUM1 were individually aligned with the corresponding region of FBF-2. The 45-residue region of FBF-2 that transfers 9-nt specificity to PUF-8 is shown in green and contains the hinge point between repeats 4 and 5. DynDom analysis also suggests that there are 2 smaller angle changes between repeats 3 and 4 and repeats 5 and 6. In C, the FBF-2 structure is rotated 15° about the y axis relative to A and B.

Fig. 5.

Small fragment of FBF opposite the flipped bases imposes its RNA-binding specificity. (A) FBF-2, with the region opposite the flipped bases, is highlighted. Helices in red are the minimal region that imposes the requirement for the extra bases relative to C. elegans PUF-8 (or PUM1). (B) Sequence alignments of FBF-1, FBF-2, and PUF-8 in the minimal region. For FBF-1, we indicate only positions that differ from the FBF-2 sequence. Helices are colored and named as in A. Yellow arrows, sites of single mutations (see the text). Identical residues are shaded in black, and similar residues are shaded in gray. (C) Binding of chimeras. Data (yeast 3-hybrid) are the average of 4 experiments, and error bars are SDs. Chimeras 1–3 used FBF-1, and chimeras 4 and 5 used FBF-2. For simplicity, we use only the FBF-2 numbering. The 2 proteins are identical in the minimal region except for residue 358. (D) In vitro binding analyses of FBF-2, PUF-8, and chimera 1 to FBE and PBE RNA. GST-fusion proteins of FBF-2, PUF-8, and chimera 1 were analyzed by electrophoretic mobility shift assay.

Fig. 6.

Conservation and adaptation of PUF protein–RNA interactions. Schematic representations of interactions between PUF proteins and their RNA targets. Interactions and RNA base conformations unique to each protein are indicated by color: FBF-2 (red), PUM1 (gold), Puf4p (green), and Puf3p (orange).