Metazoan replication-dependent histone mRNAs have a conserved stem-loop (SL) at their 3′-end. The stem–loop binding protein (SLBP) specifically recognizes the SL to regulate histone mRNA metabolism, and the 3′-5′ exonuclease 3′hExo trims its 3′-end after processing. We report the crystal structure of a ternary complex of human SLBP RNA binding domain, human 3′hExo, and a 26-nucleotide SL RNA. Only one base of the SL is recognized specifically by SLBP, and the two proteins primarily recognize the shape of the RNA. SLBP and 3′hExo have no direct contact with each other, and induced structural changes in the loop of the SL mediate their cooperative binding. The 3′ flanking sequence is positioned in the 3′hExo active site, but the ternary complex limits the extent of trimming

Dominski, Z.

Marzluff, W. F.

Tan, D.

Tong, L.

PubMed

Carolina Digital Repository

Structure of histone mRNA stem-loop, human stem-loop bindingprotein and 3′hExo ternary complex*Dazhi Tan1, William F. Marzluff2,3, Zbigniew Dominski2,3, and Liang Tong11Department of Biological Sciences, Columbia University, New York, NY 10027, USA2Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, ChapelHill, NC 27599, USA3Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill,Chapel Hill, NC 27599, USAAbstractMetazoan replication-dependent histone mRNAs have a conserved stem-loop (SL) at their 3′-end.The stem–loop binding protein (SLBP) specifically recognizes the SL to regulate histone mRNAmetabolism, and the 3′-5′ exonuclease 3′hExo trims its 3′-end after processing. We report thecrystal structure of a ternary complex of human SLBP RNA binding domain, human 3′hExo, anda 26-nucleotide SL RNA. Only one base of the SL is recognized specifically by SLBP, and thetwo proteins primarily recognize the shape of the RNA. SLBP and 3′hExo have no direct contactwith each other, and induced structural changes in the loop of the SL mediate their cooperativebinding. The 3′ flanking sequence is positioned in the 3′hExo active site, but the ternary complexlimits the extent of trimming.Metazoan replication-dependent histone mRNAs have a conserved stem-loop (SL) structureat their 3′-end. (1, 2), distinct from the polyadenylate tail found on all other knowneukaryotic mRNAs (3, 4). SLBP (5), also known as hairpin binding protein (6), is a centralregulator of histone mRNA metabolism. SLBP and the U7 snRNP (7) are required for the3′-end processing of histone pre-mRNAs (Fig. S1). SLBP is also required for the export,stability and translation of mature mRNAs. The 3′-5′ exonuclease 3′hExo (also known asEri-1) forms a tight ternary complex with SL and SLBP. 3′hExo can trim three nucleotidesin vitro from the processed histone mRNA 3′-end, and SLBP protects against furthertrimming (8-11). 3′hExo is also involved in microRNA homeostasis (12) and 5.8S rRNA3′-end maturation (13, 14). The stem-loop RNA consists of a six base-pair stem and a fourbase loop, as well as flanking sequences at both ends (Fig. S1). SLBP (31 kD) has highaffinity for the SL (Kd 1–10 nM) (15-19). It contains a ~70 residue RNA binding domain(RBD, Figs. 1A, S2). 3′hExo (40 kD) consists of an N-terminal SAP domain (~60 residues)followed by a nuclease domain (~220 residues) that belongs to the DEDDh superfamily(Figs. 1A, S3) (9-11, 20).We report here the crystal structure at 2.6 Å resolution of the ternary complex of humanSLBP RBD, human 3′hExo (SAP and nuclease domains), and a 26-nucleotide SL withconsensus sequence (Figs. 1A, 1B, Table S1) (21). Clear electron density was observed forall 26 nucleotides of the SL (Fig. 1C). The stem (nucleotides 6-11 and 16-21) has a slightly*This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to thecomplete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does notfall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS.Correspondence information for Liang Tong, Phone: (212) 854-5203, FAX: (212) 865-8246, ltong@columbia.edu.NIH Public AccessAuthor ManuscriptScience. Author manuscript; available in PMC 2013 July 18.Published in final edited form as:Science. 2013 January 18; 339(6117): 318–321. doi:10.1126/science.1228705.$watermark-text$watermark-text$watermark-textflattened classical A-form structure (Fig. S4, Table S2). Of the four nucleotides in the loop,the first (U12), second (U13) and fourth (C15) bases are flipped out (Fig. 1D). In the 3′flanking sequence, nucleotides 22-25 continue the helical structure of the stem, but the baseof the last nucleotide (A26) is flipped by ~180° relative to C25 (Fig. 1C). The riboses of allfour nucleotides in the loop (12-15) and C25 are in the 2′ endo configuration, and the RNAbackbone adopts sharp turns at these nucleotides.The structure of SLBP RBD contains three helices (αA-αC). Helices αA and αC interactwith the 5′ flanking sequence, 5′ arm of the stem and the loop of the RNA (Fig. 1B),consistent with earlier data (9, 15, 17). In particular, helix αC is positioned closest to the SLand may function as a ruler that can measure the length of the stem, with residues near its N-terminus (conserved 177-KYSRR-181 motif, Fig. S2) contacting the 5′-end of the stem andthe 5′ flanking sequence and its C-terminal region contacting the loop.The only direct recognition between SL and SLBP is through the guanine base of the secondnucleotide of the stem (G7), via two hydrogen bonds with the side-chain guanidinium groupof Arg181 (Fig. 2A). The side chain of Tyr144 (αA) is π-stacked with the first and the thirdbase, and the side chain of His195 (αC) with the fourth base of the loop (Fig. 2B). Otherinteractions are primarily between the RNA backbone and the SLBP RBD (Figs. 2C, S5).Nucleotides 3-5 in the 5′ flanking sequence, also implicated in binding to SLBP (9, 15, 17),have interactions with the RBD (Fig. S6). Besides residues Tyr178 and Ser179, theconnection between αA and αC is not in direct contact with the RNA (Fig. 1B). Thissegment contains the conserved 171-TPNK-175 sequence, and Thr171 phosphorylationproduces a 7-fold enhancement in the affinity for SL (18). This residue is located near theside chains of Lys146 (αA), Tyr151 (αA) and Trp190 (αC), and its phosphorylation mayaffect the positioning of the αA and αC helices (Fig. S6). Tyr151 is part of the conservedYDRY motif (Fig. S2), and the Y151F mutant has ~10-fold lower affinity for SL (22).3′hExo contacts the loop, the 3′ arm of the stem, and the 3′ flanking sequence of the SL(Fig. 1B), as suggested by earlier studies (9-11). The SAP domain contains three helices(α1-α3) and interacts primarily with the loop of the SL through α1 (Figs. 1B, 2B). The U13base interacts with the side chain of Tyr66 (α1) and Lys111 (α3), and the C15 base has ahydrogen bond to the side chain of Arg78 (α1). Additional interactions are with thebackbone of the RNA (Figs. 2C, S7).Nucleotides 24-26 at the 3′-end of the SL are located in the active site of the nucleasedomain of 3′hExo (Fig. 1B). The C25 base is π-stacked with that of C24 on one face andthe side chain of Trp233 on the other (Fig. 3A), thereby breaking the helical pattern of theRNA. The side chain of Arg261 is located close to the base and ribose of both C24 and C25(Fig. 3A). In comparison, the first two nucleotides of the 3′ flanking sequence (A22-C23)do not make direct contacts with 3′hExo (Fig. 3B). The binding mode of the last nucleotide(A26) is similar to that of AMP in the nuclease domain reported earlier (Fig. 3A) (20). Thephosphate group of A26 is located near the cluster of acidic side chains that coordinate twometal ions for catalysis.The crystal also contained a 3′hExo-SL binary complex (Figs. 3C, S8). SLBP RBD has lowsolubility and some of it precipitated during the preparation of the complex. The nucleasedomains of 3′hExo in the two complexes have essentially the same conformation (rmsdistance 0.4 Å). The SAP domain shows a small movement (~5° rotation), together with amovement of the RNA (Fig. 3C). However, the first eight nucleotides of the SL, includingthree at the base of the stem, are disordered in this binary complex (Fig. 3C). The structureof this binary complex is similar to that of 3′hExo in complex with a stem-loop RNAwithout any flanking sequences reported earlier (PDB entry 1ZBH), although the SAPTan et al. Page 2Science. Author manuscript; available in PMC 2013 July 18.$watermark-text$watermark-text$watermark-textdomain in that crystal comes from another 3′hExo molecule of a domain-swapped dimer(Fig. S9).Transversion of the second base pair of the stem leads to >200-fold reduction in affinity forSLBP, while transversion of the first, third, fourth or fifth base pair had <5-fold effect (17),consistent with the structural observations (Figs. 2A, S5). Mutation of Arg181 in SLBP alsoinhibited SL binding in yeast three-hybrid assays (23, 24). In comparison, transversion ofthe second base pair had little effect on 3′hExo binding (9), also consistent with thestructure (Table S3). To further validate the structural observations, we introduced mutationsin the SL-SLBP RBD and SL-3′hExo interfaces and determined their effects on theformation of the binary and ternary complexes. Overall, the mutagenesis results are in goodagreement with the structure (Fig. S10, Table S4).The modes of SL recognition by the RBD of SLBP and the SAP domain of 3′hExo appearto be distinct from other RNA binding proteins. The SLBP RBD does not have a closestructural homolog in the PDB. While the SAP domain has structural similarity to a domainin the recombination endonuclease VII (25), it does not share a common mode of nucleicacid interaction with that domain.Our studies suggest that SLBP RBD and 3′hExo recognize the overall shape of the SL(especially its loop) rather than the sequence, which is also supported by observations fromthe single transversion studies (9, 17). At the same time, the sequence of the SL plays a rolein determining its shape. The first (U12) and third (U14) nucleotides of the loop are highlyconserved (Fig. S11) and contribute to the specificity of recognition (9, 17). C. elegansSLBP is more selective for a C at the first position of the loop, while human SLBP bindsRNAs with C or U at the first position with comparable affinity (16). Tyr144 of humanSLBP is replaced by an Arg residue in C. elegans SLBP, and this may result in a distinctmechanism of recognizing the C in the first nucleotide of the loop (Fig. 2B).There are no direct contacts between SLBP RBD and 3′hExo in the ternary complex (Figs.1B, S12). The RBD and SAP domain are arranged on opposite sides of the loop, and theyapproach each other most closely there. Cooperative binding between the two proteins(9-11) is likely due to induced structural changes in the loop, such that binding of oneprotein induces a conformation of the loop that promotes the binding of the other protein. Instructures of the SL alone in solution (26, 27), the conformation of the loop region isdifferent from that in the complex observed here (Fig. S13).3′hExo has primarily bipartite interactions with the SL. The SAP domain recognizes theloop while the nuclease domain binds the 3′ flanking sequence (Fig. 1B). Disruption ofinteractions at either of these two sites leads to reduced binding (9, 10). Nucleotide A26would be the leaving group for the 3′-5′ exonuclease activity (Fig. 3A), which does notshow sequence preference (9) as neither C25 nor A26 is recognized specifically. While3′hExo can remove the last 3 nucleotides of the SL (9), further degradation is not possibleas the 3′-end of the shortened SL can no longer reach the active site of 3′hExo in the ternarycomplex (Fig. 3B), thereby explaining how SLBP protects histone mRNAs from excessivetrimming by 3′hExo.Besides recognizing the SL RNA, another function of SLBP is the recruitment of U7 snRNPand stabilization of its interaction with the histone pre-mRNA for 3′-end processing (Fig.S1) (22, 28). The 20 residues immediately C-terminal to the RBD of SLBP are required forthis processing (28). These residues are present in the recombinant SLBP used in the currentstructural studies, but they are disordered. A second region required for processing is locatedin helix αB of the RBD, especially the YDRY motif (Figs. 1B, S6) where mutation of theDR residues to QC did not affect binding but abolished processing (22). Our structure showsTan et al. Page 3Science. Author manuscript; available in PMC 2013 July 18.$watermark-text$watermark-text$watermark-textthat these two regions are likely located close to each other (Fig. S6), and therefore alsoidentifies a surface feature of SLBP that is involved in histone pre-mRNA 3′-end processing(Fig. S14).Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe thank Neil Whalen, Stuart Myers, Rick Jackimowicz and Howard Robinson for access to the X29A beamline atthe NSLS. This research was supported in part by grants from the NIH to LT (GM077175) and to WFM and ZD(GM029832). The structure has been deposited at the PDB (accession code 4HXH).References1. Dominski Z, Marzluff WF. Gene. 2007; 396:373. [PubMed: 17531405]2. Marzluff WF, Wagner EJ, Duronio RJ. Nat. Rev. Genet. 2008; 9:843. [PubMed: 18927579]3. Zhao J, Hyman L, Moore CL. Microbiol. Mol. Biol. Rev. 1999; 63:405. [PubMed: 10357856]4. Mandel CR, Bai Y, Tong L. Cell. Mol. Life Sci. 2008; 65:1099. [PubMed: 18158581]5. Wang ZF, Whitfield ML, Ingledue TC III, Dominski Z, Marzluff WF. Genes Develop. 1996;10:3028. [PubMed: 8957003]6. Martin F, Schaller A, Eglite S, Schumperli D, Muller B. EMBO J. 1997; 16:769. [PubMed:9049306]7. Mowry KL, Steitz JA. Science. 1987; 238:1682. [PubMed: 2825355]8. Mullen TE, Marzluff WF. Genes Develop. 2008; 22:50. [PubMed: 18172165]9. Dominski Z, Yang X-C, Kaygun H, Dadlez M, Marzluff WF. Mol. Cell. 2003; 12:295. [PubMed:14536070]10. Yang X-C, Purdy M, Marzluff WF, Dominski Z. J. Biol. Chem. 2006; 281:30447. [PubMed:16912046]11. Yang X-C, Torres MP, Marzluff WF, Dominski Z. Mol. Cell. Biol. 2009; 29:4045. [PubMed:19470752]12. Thomas MF, et al. Blood. 2012; 120:130. [PubMed: 22613798]13. Ansel KM, et al. Nature Struct. Mol. Biol. 2008; 15:523. [PubMed: 18438418]14. Gabel HW, Ruvkun G. Nature Struct. Mol. Biol. 2008; 15:531. [PubMed: 18438419]15. Williams AS, Marzluff WF. Nucl. Acid Res. 1995; 23:654.16. Michel F, Schumperli D, Muller B. RNA. 2000; 6:1539. [PubMed: 11105754]17. Battle DJ, Doudna JA. RNA. 2001; 7:123. [PubMed: 11214174]18. Borchers CH, et al. Proc. Natl. Acad. Sci. USA. 2006; 103:3094. [PubMed: 16492733]19. Zhang M, Lam TT, Tonelli M, Marzluff WF, Thapar R. Biochem. 2012; 51:3215. [PubMed:22439849]20. Cheng Y, Patel DJ. J. Mol. Biol. 2004; 343:305. [PubMed: 15451662]21. Detailed materials and methods can be found in the supplementary information.22. Dominski Z, Erkmann JA, Greenland JA, Marzluff WF. Mol. Cell. Biol. 2001; 21:2008. [PubMed:11238936]23. Martin F, Michel F, Zenklusen D, Muller B, Schumperli D. Nucl. Acid Res. 2000; 28:1594.24. Jaeger S, Eriani G, Martin F. FEBS Lett. 2004; 556:265. [PubMed: 14706861]25. Biertumpfel C, Yang W, Suck D. Nature. 2007; 449:616. [PubMed: 17873859]26. DeJong ES, Marzluff WF, Nikonowicz EP. RNA. 2002; 8:83. [PubMed: 11871662]27. Zanier K, et al. RNA. 2002; 8:29. [PubMed: 11871659]28. Dominski Z, Zheng LX, Sanchez R, Marzluff WF. Mol. Cell. Biol. 1999; 19:3561. [PubMed:10207079]Tan et al. Page 4Science. Author manuscript; available in PMC 2013 July 18.$watermark-text$watermark-text$watermark-textFigure 1.Structure of human SLBP RBD, human 3′hExo, and SL RNA ternary complex. (A).Domain organizations of human SLBP and human 3′hExo. Residues not included in theexpression constructs are shown in gray. (B). Schematic drawings of the structure of theternary complex of human SLBP RBD (cyan), human 3′hExo (SAP domain in yellow andnuclease domain in green), and 26-nucleotide SL RNA (orange). (C). Simulated annealingomit Fo–Fc electron density (light green) for the SL RNA at 2.6 Å resolution, contoured at3σ. (D). Closeup of the loop region of the SL RNA. All the structure figures were producedwith PyMOL (www.pymol.org).Tan et al. Page 5Science. Author manuscript; available in PMC 2013 July 18.$watermark-text$watermark-text$watermark-textFigure 2.Interactions between the SL RNA and SLBP RBD and 3′hExo. (A). Specific recognition ofG7 in the second base pair of the stem (orange) by hydrogen bonding (dashed lines in red)with the side chain of Arg181 (cyan) of SLBP. Simulated annealing omit Fo–Fc electrondensity for G7 and Arg181 is also shown, contoured at 5σ. (B). Interactions between theloop of the SL RNA (orange) with the SLBP RBD (cyan) and the 3′hExo SAP domain(yellow). (C). Schematic drawing summarizing the interactions between SL and SLBP RBD(cyan) and 3′hExo.Tan et al. Page 6Science. Author manuscript; available in PMC 2013 July 18.$watermark-text$watermark-text$watermark-textFigure 3.The 3′ flanking sequence of the SL RNA is located in the 3′hExo active site. (A).Interactions between the 3′ flanking sequence of the SL RNA (orange) with the active siteof 3′hExo nuclease domain (green). The bound positions of AMP (gray) and two metal ions(pink spheres) to the nuclease domain of 3′hExo as observed earlier are also shown (20).(B). Molecular surface of the active site region of 3′hExo colored based on electrostaticpotential. The SL RNA is shown as a stick model (orange). The black arrow indicatesanother opening from the active site, through which 3′hExo may accommodate longer RNAmolecules. (C). Overlay of the structures of the ternary (SL-SLBP-3′hExo, in color) andbinary (gray for 3′hExo and black for SL) complexes. The superposition is based on thenuclease domain of 3′hExo.Tan et al. Page 7Science. Author manuscript; available in PMC 2013 July 18.$watermark-text$watermark-text$watermark-text

Structure of Histone mRNA Stem-Loop, Human Stem-Loop Binding Protein, and 3'hExo Ternary Complex

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Structure of Histone mRNA Stem-Loop, Human Stem-Loop Binding Protein, and 3'hExo Ternary Complex

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