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Structural Basis for Specific Recognition of Multiple Mrna Targets by a PUF Regulatory Protein

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Structural Basis for Specific Recognition of Multiple Mrna Targets by a PUF Regulatory Protein Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein Yeming Wanga, Laura Oppermanb, Marvin Wickensb,1, and Traci M. Tanaka Halla,1 aLaboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; and bDepartment of Biochemistry, University of Wisconsin, Madison, WI 53706 Edited by Keith R. Yamamoto, University of California, San Francisco, CA, and approved October 2, 2009 (received for review December 2, 2008) Caenorhabditis elegans fem-3 binding factor (FBF) is a founding association in worm extracts, immunocytochemistry, and genetics member of the PUMILIO/FBF (PUF) family of mRNA regulatory (5, 8, 23–27). FBF is required for maintenance of germline stem proteins. It regulates multiple mRNAs critical for stem cell mainte- cells, a conserved function of PUF proteins (1). FBF maintains stem nance and germline development. Here, we report crystal struc- cells by repressing the expression of differentiation regulators, tures of FBF in complex with 6 different 9-nt RNA sequences, including gld-1, fog-1, lip-1, and mpk-1 (28). In addition, FBF including elements from 4 natural mRNAs. These structures reveal regulates the switch from spermatogenesis to oogenesis by repress- that FBF binds to conserved bases at positions 1–3 and 7–8. The key ing fem-3 mRNA (8). The regulatory elements found in the specificity determinant of FBF vs. other PUF proteins lies in posi- different mRNAs are related, but distinct. PUF proteins coimmu- tions 4–6. In FBF/RNA complexes, these bases stack directly with noprecipitate with many mRNAs in yeast, fly, and human extracts one another and turn away from the RNA-binding surface. A short (4, 29, 30). Although the biological relevance of all the interactions region of FBF is sufficient to impart its unique specificity and lies is not yet clear, the conclusion that a single PUF protein associates directly opposite the flipped bases. We suggest that this region with a large number of functionally related mRNAs is inescapable. imposes a flattened curvature on the protein; hence, the require- The biochemical basis of FBF’s specificity for RNA has been ment for the additional nucleotide. The principles of FBF/RNA examined in depth and provides a foundation for structural analysis. recognition suggest a general mechanism by which PUF proteins FBF binds a 9-nt sequence, despite having only 8 PUF repeats (20). recognize distinct families of RNAs yet exploit very nearly identical The core FBF recognition sequence begins with a UGU triplet, as atomic contacts in doing so. do all validated PUF target sequences. The optimal binding site is 5Ј-UGURNNAUA-3Ј (R, purine; N, any base; see Results) (28). crystal structure ͉ RNA ͉ translational regulation ͉ fem-3 binding factor ͉ PUF-8, a close relative in C. elegans to PUMILIO, recognizes a core base flipping 8-nt sequence (20), 5Ј-UGUANAUA-3Ј (4, 30–32), the same as that seen by PUM1 (30). Biochemistry and genetics identified the RNA regulation permeates biology. Elements in 3Ј-UTRs are fifth position of the FBF recognition sequence as an inserted base mrecognized by regulatory proteins that activate, repress, sta- relative to the PUF-8 (20). It has been proposed that a structural bilize, and destroy target mRNAs. Their specificity defines which distortion in the central region of the protein imposes a requirement mRNAs are regulated, and so determines biological outcomes. for a ‘‘spacer’’ nucleotide (20). This ‘‘extra’’ base is an identity PUMILIO/fem-3 binding factor (FBF) proteins (1, 2), or PUF determinant that enables the protein to bind and regulate cognate proteins, are exemplary and regulate batteries of mRNAs to control mRNAs uniquely. stem cell maintenance and memory (1, 3–5). Understanding the We sought to understand how FBF specifically recognizes its 9-nt structural basis of their RNA-binding specificities is a critical goal. mRNA targets despite having 8 repeats. To do so, we determined The PUF protein family includes members throughout eu- crystal structures of the RNA-binding domain of FBF-2 and 6 karyotes. To date, PUF proteins invariably bind to elements in different RNA sequences, including 4 natural target sequences. The 3Ј-UTRs (1). In most instances, the PUF/RNA complexes decrease data not only reveal the basis of FBF specificity but lead to general expression of the mRNA, although activation also can occur (6, 7). models of PUF protein RNA specificity and their evolution. PUF proteins contain a conserved RNA-binding domain, known as the Pumilio-homology domain or PUF domain. This domain Results comprises 8 PUF repeats, flanked by pseudorepeats (8–11). The Crystal Structures of FBF-2 in Complex with 9-nt RNA Target Se- PUF domain is sufficient to bind target mRNAs (8, 10, 12), recruit quences. A crystal structure of the RNA-binding domain of FBF-2 partner proteins (13–16), and regulate mRNAs in vivo (2, 17). in complex with a 9-nt consensus FBF binding element (FBE), A typical PUF repeat contains 3 ␣-helices, and the 8 repeats in 5Ј-UGUACUAUA-3Ј (20, 28), was determined by single wave- a single protein pack to form a crescent shape (16, 18). In human length anomalous diffraction (SAD) and refined to a resolution of PUMILIO1 (PUM1) bound to a nanos response element (NRE) 2.2 Å. The initial experimental electron density map revealed RNA, each PUF repeat interacts with a single RNA base along an density for all 9 RNA bases [supporting information (SI) Fig. S1]. extended concave surface (19). In a typical PUF repeat, the second As a convention, we designate the 5Ј-UGU as positions 1–3 in the ␣-helix provides 3 conserved amino acids that interact with an RNA recognition sequences. base. Two residues contact the edge of the base via hydrogen bonding or van der Waals interactions, and a third often is sandwiched between 2 bases to form stacking interactions. The Author contributions: Y.W., L.O., M.W., and T.M.T.H. designed research; Y.W. and L.O. performed research; Y.W., L.O., M.W., and T.M.T.H. analyzed data; and Y.W., L.O., M.W., identities of the residues contacting the edge of the bases play a key and T.M.T.H. wrote the paper. role in selecting the RNA base (19–22). The authors declare no conflict of interest. The Caenorhabditis elegans PUF proteins, FBF-1 and FBF-2, This article is a PNAS Direct Submission. provide a special opportunity to dissect the structural basis of Data deposition: The atomic coordinates and structure factors have been deposited in the PUF–RNA interactions in a biological context. FBF-1 and FBF-2 Protein Data Bank, www.pdb.org (PDB ID codes 3K5Q, 3K5Y, 3K5Z, 3K61, 3K62 and 3K64). are 91% identical in amino acid sequence, have similar RNA- 1To whom correspondence may be addressed. E-mail: [email protected] or binding specificity, and overlap functionally (8, 23–27); they are [email protected]. referred to collectively as FBF. Eight mRNA targets of FBF have This article contains supporting information online at www.pnas.org/cgi/content/full/ been identified using a combination of in vitro binding, physical 0812076106/DCSupplemental. 20186–20191 ͉ PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812076106 Downloaded by guest on October 1, 2021 R8 R8 U1 Q504 A B A (Q1126) B H326 R7 R7 U1 (H972) C C G2 R8´ R8´ N500 R4 (N1122) R6 R6 U3 A7 5´ 5´ Y501 (A6) Q291 G4 (Y1123) (Q939) G2 R8 R5 R5 C287 C5 E457 R288 (R936) (C935) C6 (E1083) R4 R4 H454 U8 A7 (N1080) S453 (U7) R3 3´ R3 3´ R3 (S1079) Y245 Q248 U3 (Y900) (Q903) R2 R2 N244 U8 R7 (N899) N N Q419 R1 R1 A9 (Q1047) R1´ R1´ R6 A9 G4 (A8) (Q867) (A4) N415 K201 R2 (N1043) (Q860) Fig. 1. Crystal structure of FBF-2 in complex with gld-1 FBEa RNA. (A) (R864) C204 Y416 (S863) Stereo-view of FBF-2 in complex with gld-1 FBEa RNA. Repeats are colored (Y1044) R1 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; Fig. 2. Recognition of conserved RNA regions by FBF-2. (A) Conservation of orange, phosphorus; yellow, sulfur). Bases 4–6 are shown with green carbon PUF protein recognition of the 5Ј-conserved UGU sequence. Superposition of atoms. (B) Surface representation of FBF-2 in complex with gld-1 FBEa RNA. repeats 6–8 in the structures of FBF-2 in complex with gld-1 FBEa RNA and The figures were prepared with PyMol (44). 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 We subsequently determined the crystal structures of FBF-2 in repeats 6–8 of FBF-2 and PUM1 were aligned (rmsd of 1.41 Å over 105 CA complex with 9-nt sequences from the 3Ј-UTRs of natural mRNA atoms). The RNA-interacting side chains of PUM1 are labeled in parentheses. targets: the gld-1 FBEa and FBEb mediate repression of gld-1 (B) Conservation of PUF protein recognition at the 3Ј-end. Corresponding CA mRNA to control stem cells (23); the fog-1 FBEa triggers repression atoms in repeats 1–3 of FBF-2 and PUM1 were aligned (rmsd of 1.70 Å over 99 CA atoms). to permit oogenesis (27); and the fem-3 point mutation element (PME) mediates repression to permit the switch from spermato- BIOCHEMISTRY genesis to oogenesis (27).
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