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Structural Organization of Box C/D RNA-Guided RNA Methyltransferase

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Structural Organization of Box C/D RNA-Guided RNA Methyltransferase Structural organization of box C/D RNA-guided RNA methyltransferase Keqiong Yea,1, Ru Jiaa, Jinzhong Lina, Minghua Jua, Jin Penga, Anbi Xua, and Liman Zhanga,b aNational Institute of Biological Sciences, Beijing 102206, China; and bGraduate Program at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China Edited by Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved June 29, 2009 (received for review May 11, 2009) Box C/D guide RNAs are abundant noncoding RNAs that primarily In archaea, C/D RNA associates with 3 proteins—fibrillarin, function to direct the 2؅-O-methylation of specific nucleotides by Nop5 (also known as Nop56/58), and L7Ae—to form an enzyme base-pairing with substrate RNAs. In archaea, a bipartite C/D RNA capable of catalyzing site-specific modification (17). In eukary- assembles with L7Ae, Nop5, and the methyltransferase fibrillarin otic complex, paralogs Nop56 and Nop58 replace the single into a modification enzyme with unique substrate specificity. Here, Nop5 protein, and a 15.5-kDa protein (15.5K, Snu13 in yeast) is we determined the crystal structure of an archaeal C/D RNA– equivalent to L7Ae. Fibrillarin is the methyltransferase, and it protein complex (RNP) composed of all 3 core proteins and an catalyzes the transfer of a methyl group from bound S-adenosyl- engineered half-guide RNA at 4 Å resolution, as well as 2 protein methionine (SAM) to the 2Ј-hydroxyl group of the target ribose substructures at higher resolution. The RNP structure reveals that (17–19). L7Ae/15.5K is a primary RNA-binding protein that the C-terminal domains of Nop5 in the dimeric complex provide recognizes the K-turn (10, 11, 13). L7Ae also binds well to the symmetric anchoring sites for 2 L7Ae-associated kink-turn motifs of K-loop (20, 21), but eukaryotic 15.5K does not (21–23). Nop5 is the C/D RNA. A prominent protrusion in Nop5 seems to be impor- a scaffold protein composed of an N-terminal domain (NTD) tant for guide RNA organization and function and for discriminat- that binds fibrillarin, a coiled-coil domain that mediates self- ing the structurally related U4 snRNA. Multiple conformations of dimerization, and a C-terminal domain (CTD, also known as the the N-terminal domain of Nop5 and its associated fibrillarin in Nop domain) that likely binds the L7Ae–RNA complex (24). different structures indicate the inherent flexibility of the catalytic C/D RNP assembly proceeds in a stepwise manner (17, 20, 24, module, suggesting that a swinging motion of the catalytic module 25) and begins with the formation of the L7Ae–C/D RNA is part of the enzyme mechanism. We also built a model of a native complex. Nop5 associates with the preassembled L7Ae–C/D C/D RNP with substrate and fibrillarin in an active conformation. RNP but cannot stably bind C/D RNA or L7Ae alone. Fibrillarin Our results provide insight into the overall organization and is recruited through its interaction with Nop5. mechanism of action of C/D RNA–guided RNA methyltransferases. The structure of archaeal C/D RNP has a 2-fold symmetry, with structurally equivalent C/D and CЈ/DЈ motifs in RNA, 2 crystal structure ͉ methylation ͉ RNA–protein complex ͉ non-coding RNA copies of each protein, and self-association of Nop5. The integ- rity of the symmetric structure is important for the activity (20, ore than 100 types of chemical modification are intro- 26–28), particularly for the substrate specificity (29). By contrast, duced at specific sites of cellular RNAs after their tran- the structure of the eukaryotic C/D RNP is less symmetric at M Ј Ј scription (1). These modifications, which greatly increase the both the RNA and protein levels. The eukaryotic C and D chemical diversity of RNA, are generally beneficial and some- boxes are frequently divergent from the consensus sequence times even critical for the structure and function of host RNAs. required for K-turn formation and cannot bind 15.5K in isolation RNA modifications are normally carried out by individual (21–23). Nop58 and Nop56 preferentially bind box C/D and box Ј Ј protein enzymes. However, an exception is the large number of C /D , respectively (30). pseudouridine and 2Ј-O-methylated nucleotides in rRNAs, snR- Crystal structures have been solved for fibrillarin alone (19), NAs, and tRNAs that are synthesized by H/ACA and C/D 2 Nop5–fibrillarin complexes [from Archaeoglobus fulgidus (AF) RNA–protein complexes (RNPs), respectively (2–6). In each and Pyrococcus furiosus (PF)] (24, 31), and a number of L7Ae/ such complex, a distinct guide RNA belonging to the H/ACA or 15.5K and K-turn/K-loop RNA complexes (11, 13, 16, 32). The C/D RNA family determines the substrate specificity by base- spliceosomal U4 small nuclear RNP (snRNP) shares 3 homol- Ј pairing with the substrate around the modification site. These 2 ogous components with the C/D RNP: a K-turn in the 5 stem Ј classes of RNA-guided RNA modification enzymes are ubiqui- loop (5 SL) of the U4 snRNA, 15.5K, and the Nop5 homolog tous in eukaryotic and archaeal organisms but are not found in Prp31. The crystal structure of a ternary U4 snRNP complex bacteria. revealed that the CTD of Prp31 is an RNP binding domain The methylation guide C/D RNAs contain a C box (RUG- recognizing a composite surface formed by the K-turn and 15.5K AUAG, R is purine) near the 5Ј end and a D box (CUGA) near (33). The ternary U4 snRNP structure provides a model for the 3Ј end, and related boxes CЈ and DЈ at the internal region. understanding C/D RNP assembly, yet is insufficient to explain One or 2 antisense sequences are present upstream of boxes D the unique guide function of the C/D RNA. Despite these and DЈ and form a 10–21-bp duplex with the substrate, thereby advances, the structure of a fully assembled C/D RNP is not yet selecting the nucleotide paired to the fifth nucleotide upstream of box D/DЈ for modification (7–9). Boxes C and D combine and Author contributions: K.Y., R.J., and J.L. designed research; K.Y., R.J., J.L., M.J., J.P., A.X., and fold into a kink-turn (K-turn) structural motif (10–13). A L.Z. performed research; K.Y., R.J., and J.L. analyzed data; and K.Y. wrote the paper. canonical K-turn is composed of 2 bent stems linked by a 3-nt The authors declare no conflict of interest. bulge with an extruded nucleotide. One stem contains tandem This article is a PNAS Direct Submission. sheared G⅐A pairs (hereafter called the GA-stem, also known as Data deposition: Data deposition: The atomic coordinates and structure factors have been stem II or noncanonical stem), and the other contains Watson– deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 3ID5 (C/D RNP structure), Crick pairs (WC-stem, also known as stem I or canonical stem). 3ICX (Nop5⌬NTD structure), and 3ID6 (Nop5⌬CTD-Fib complex structure)]. In archaeal C/D RNAs (14), the internal CЈ and DЈ boxes often 1To whom correspondence should be addressed. E-mail: [email protected]. form a related K-loop structure, in which a loop replaces the This article contains supporting information online at www.pnas.org/cgi/content/full/ WC-stem in the canonical K-turn (15, 16). 0905128106/DCSupplemental. 13808–13813 ͉ PNAS ͉ August 18, 2009 ͉ vol. 106 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905128106 Downloaded by guest on September 23, 2021 Fig. 2. The structures of Nop5 and fibrillarin. (A) The structure of dimeric Nop5⌬NTD. One subunit is colored deep blue, and the other is colored according to individual domains, with the CTD in red, the coiled-coil domain in magenta, and the tip insertion domain in orange. Dots strand for disordered ⌬ Fig. 1. Natural and half-C/D RNAs. (A) Schematic of the bipartite structure of loops. (B) Structure of the Nop5 CTD–Fib complex. Only a single copy is an archaeal C/D RNA. The terminal boxes C and D form a canonical K-turn, shown. The coiled-coil domain has the same orientation as in Fig. 2A. The CTD whereas the internal boxes CЈ and DЈ fold into a K-loop. Boxes C/CЈ and D/DЈ and NTD of Nop5 are colored dark and light green, respectively, and fibrillarin are represented by their consensus sequences surrounded by red boxes. The (Fib) is indicated in cyan. (C) The SS and PF Nop5 NTD–Fib complex structures guide upstream of box D (D-guide) is shown to associate with the substrate aligned by fibrillarin. (D) Variable orientation between the NTD and the ⌬ ⌬ (gray), whereas the guide upstream of box DЈ (DЈ-guide) is unbound. The coiled-coil domain of Nop5. Nop5 structures in the SS Nop5 CTD–Fib (SS- C, hollow red circle strands for the modification target that is paired with the 5th yellow), SS C/D RNP (SS-RNP, green), PF Nop5–Fib (PF, magenta), and AF nucleotide upstream of box D. Arrows indicate the cutting sites for generating Nop5–Fib (AF, blue) complexes, as well as an active model (active-like, gray), ␣ the half-C/D RNA. (B) The sequence and secondary structure of the half-C/D are shown. Only the 3 helix of the Nop5 NTD is shown for clarity. These RNA used in crystallization.
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