title: Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots.
abstract: Caenorhabditis elegans CPB-1 (cytoplasmic polyadenylation element binding protein homolog-1) and FBF (fem-3 mRNA binding factor) are evolutionary conserved regulators of mRNA translation that belong to the CPEB (cytoplasmic polyadenylation element binding) and PUF (Pumilio and FBF) protein families, respectively. In hermaphrodite worms, CPB-1 and FBF control key steps during germline development, including stem cell maintenance and sex determination. While CPB-1 and FBF are known to interact, the molecular basis and function of the CPB-1⋅FBF complex are not known. The surface of CPB-1 that interacts with FBF was localized using in vivo and in vitro methods to a 10-residue region at the N-terminus of the protein and these residues are present in the FBF-binding protein GLD-3 (germline development defective-3). PUF proteins are characterized by the presence of eight α-helical repeats (PUF repeats) arranged side by side in an elongated structure. Critical residues for CPB-1 binding are found in the extended loop that connects PUF repeats 7 and 8. The same FBF residues also mediate binding to GLD-3, indicating a conserved binding mode between different protein partners. CPB-1 binding was competitive with GLD-3, suggestive of mutual exclusivity in vivo. RNA binding measurements demonstrated that CPB-1 alters the affinity of FBF for specific RNA sequences, implying a functional model where the coregulatory protein CPB-1 modulates FBF target selection.
authors.affiliation: Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Menichelli
authors.forename: Joann
authors.initials: J
authors.lastname: Wu
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Campbell
authors.forename: Marvin
authors.initials: M
authors.lastname: Wickens
authors.forename: James R
authors.initials: JR
authors.lastname: Williamson
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D005786:Gene Expression Regulation; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D000596:Amino Acids; C416182:CPB-1 protein, C elegans; D029742:Caenorhabditis elegans Proteins; C469731:GLD-3 protein, C elegans; D016601:RNA-Binding Proteins; C109286:fem-3-binding protein, C elegans; D012313:RNA