Structural organization of box C/D RNA-guided RNA methyltransferase

Ye, Keqiong; Jia, Rong; Lin, Jinzhong; Ju, Minghua; Jin, Peng; Xu, Anbi; Zhang, Liman

doi:10.1073/pnas.0905128106

Cited by 60 publications

(100 citation statements)

References 42 publications

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“…Rrp9 may simultaneously recognize the B/C motif and Snu13 in the context of U3 snoRNP. Such a binding mode would be similar to that adopted by Nop5, which binds both L7Ae and the K-turn in the archaeal box C/D RNP (Ye et al 2009;Xue et al 2010;Lin et al 2011). In support of this model, a weak direct or GFP-fusions with mutant Rrp9 that contains deletion (Δ) of residues 1-55 (g), 56-80 (h), 1-80 (i), 1-126 ( j), the 7bc loop (residues 547-552) (k), or both residues 1-126 and the 7bc loop (l ).…”

Section: Discussionmentioning

confidence: 99%

Structural and functional analysis of the U3 snoRNA binding protein Rrp9

Zhang

Lin

2013

RNA

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The U3 snoRNA is required for 18S rRNA processing and small subunit ribosome formation in eukaryotes. Different from other box C/D snoRNAs, U3 contains an extra 5 ′ domain that pairs with pre-rRNA and a unique B/C motif essential for recruitment of the U3-specific Rrp9 protein. Here, we analyze the structure and function of Rrp9 with crystallographic, biochemical, and cellular approaches. Rrp9 is composed of a WD repeat domain and an N-terminal region. The crystal structures of the WD domain of yeast Rrp9 and its human ortholog U3-55K were determined, revealing a typical seven-bladed propeller fold. Several conserved surface patches on the WD domain were identified, and their function in RNP assembly and yeast growth were analyzed by mutagenesis. Prior association of Snu13 with the B/C motif was found to enhance the specific binding of the WD domain. We show that a conserved 7bc loop is crucial for specific recognition of U3, nucleolar localization of Rrp9, and yeast growth. In addition, we show that the N-terminal region of Rrp9 contains a bipartite nuclear localization signal that is dispensable for nucleolar localization. Our results provide insight into the functional sites of Rrp9.

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Section: Discussionmentioning

confidence: 99%

Structural and functional analysis of the U3 snoRNA binding protein Rrp9

Zhang

Lin

2013

RNA

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“…26 It has been observed that the K-turn sRNA would have two distinct tertiary structures, e.g. it has plasticity to some extent.…”

Section: Introductionmentioning

confidence: 99%

Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations

Wei¹,

Yang²,

Yu³

et al. 2013

Phys. Chem. Chem. Phys.

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“…Purification of Nop5, fibrillarin, and L7Ae proteins; preparation of C/D RNAs; and assembly of C/D RNPs were done as described previously (29,30). Crystallization was performed by the hanging-drop vapor diffusion method at 20°C.…”

Section: Discussionmentioning

confidence: 99%

“…Nop5 dimerizes through its central coiled-coil domain and binds fibrillarin with its N-terminal domain (NTD) (27). The K-turn/K-loop structure of C/D RNA is sandwiched between L7Ae and the C-terminal domain (CTD) of Nop5 (28,29). The linker between the NTD and the coiled-coil domain of Nop5 is flexible, allowing fibrillarin to access the bound substrate in a dynamic manner (27,(29)(30)(31).…”

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confidence: 99%

“…The K-turn/K-loop structure of C/D RNA is sandwiched between L7Ae and the C-terminal domain (CTD) of Nop5 (28,29). The linker between the NTD and the coiled-coil domain of Nop5 is flexible, allowing fibrillarin to access the bound substrate in a dynamic manner (27,(29)(30)(31).We have previously determined a crystal structure of substratebound C/D RNP in the active conformation (30). The structure illustrates the organization of a monomeric C/D RNP (mono-RNP), in which a single bipartite C/D RNA is anchored by its two K-turn motifs onto the Nop5 dimer platform.…”

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confidence: 99%

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Box C/D guide RNAs recognize a maximum of 10 nt of substrates

Yang

Lin

2016

Proc. Natl. Acad. Sci. U.S.A.

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Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide-substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with <12 nucleotides are defective, mainly because they cannot load the substrate in the active conformation. For spacers with >12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNAswapped model for dimeric C/D RNP.B ox C/D RNAs are a large family of noncoding RNAs conserved in archaea and eukaryotes (1-3). The majority of these RNAs function as guides in site-specific 2′-O-methylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (4-7). A few special members, including U3 and U14, small nucleolar RNAs are required for pre-rRNA processing and ribosome assembly. One of the most abundant modifications in rRNAs, 2′-O-methylated nucleotides cluster in functionally important regions and are believed to fine-tune ribosome structure and function (8)(9)(10)(11).Box C/D RNAs have a bipartite structure and contain the C (RUGAUGA; R is a purine) and D (CUGA) box motifs at two ends and the related C′ and D′ box motifs in the internal regions. Each pair of boxes C and D combine into a characteristic kink turn (K-turn) or K-loop structure (12-15). The spacer regions located between boxes D and C′ (D spacer) and between boxes D′ and C (D′ spacer) harbor the guide sequence. The guide sequence can potentially form 10-21 bp with complementary sequences in substrate RNAs and select the fifth nucleotide apart from box D′/D for modification (5).To form a functional ribonucleoprotein (RNP) enzyme, each C/D RNA associates with four core proteins [Snu13, fibrillarin (Nop1 in yeast), Nop56, and Nop58] in eukaryotes (16-23) and with three proteins (L7Ae, fibrillarin, and Nop5) in archaea (24). The homologous eukaryotic Nop56 and Nop58 proteins are replaced by a single protein, Nop5, in archaea. Fibrillarin is the catalyst that transfers a methyl group from S-adenosylmethionine to the target ribose (21, 25). The RNA-guided methylation activity has been reconstituted for archaeal C/D RNPs (24), but not yet for eukaryotic complexes (26).Structural st...

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Structural organization of box C/D RNA-guided RNA methyltransferase

Cited by 60 publications

References 42 publications

Structural and functional analysis of the U3 snoRNA binding protein Rrp9

Structural and functional analysis of the U3 snoRNA binding protein Rrp9

Kink turn sRNA folding upon L7Ae binding using molecular dynamics simulations

Box C/D guide RNAs recognize a maximum of 10 nt of substrates

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