Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Lin, Jinzhong; Lai, Shaomei; Jia, Rong; Xu, Anbi; Zhang, Liman; Lü, Jing; Ye, Keqiong

doi:10.1038/nature09688

Cited by 120 publications

(188 citation statements)

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“…S1 B and C). A similar arrangement of two antiparallel coiled-coil domains is observed in the structures of box C/D RNA protein complexes (32). In these complexes, the coiled-coil domains play an important scaffolding role, and are required for correct positioning of other domains and interacting proteins for binding and modification of RNA.…”

Section: Resultsmentioning

confidence: 71%

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Williams

Catanzariti

et al. 2013

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

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Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system. innate immunity | plant cell internalization | plant disease resistance | avirulence protein | lipid binding F ilamentous eukaryotic microbes such as fungi and oomycetes cause devastating diseases in many economically important crop plants, including rice, corn, wheat, soybean, and potato. During infection, oomycetes and biotrophic fungal pathogens establish a physical interaction with their host through specialized feeding structures, known as haustoria, which facilitate secretion of a vast array of effector proteins to overcome plant defenses and promote host colonization (1-3). The effectors are generally highly divergent even among related species, and generally lack similarity to proteins currently available in the databases, making it difficult to predict biological function from sequence alone. Some of the effectors accumulate in the plant intercellular space (apoplast), whereas others are translocated into the host cell. At present, the host targets and virulence mechanisms of fungal and oomycete effectors are poorly understood. A subset of the planttranslocated effectors called avirulence (Avr) proteins are recognized directly or indirectly and with high specificity by plant disease resistance (R) proteins (4-6). This recognition event leads to activation of effector-triggered immunity (ETI), which usually includes rapid localized host cell death at the site of infectiontermed the hypersensitive response (HR)-and results in the plants being immune to pathogen infection.The question of how...

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Section: Resultsmentioning

confidence: 71%

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Williams

Catanzariti

et al. 2013

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

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“…In vitro reconstitution of archaeal C/D RNPs also yielded a dimeric RNP (di-RNP) (30,34). The structural organization and biological relevance of di-RNP is currently a matter of debate.…”

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confidence: 99%

“…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%

“…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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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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Nop5 interacts with the archaeal RNA exosome

et al. 2017

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The archaeal exosome, a protein complex responsible for phosphorolytic degradation and tailing of RNA, has an RNA-binding platform containing Rrp4, Csl4, and DnaG. Aiming to detect novel interaction partners of the exosome, we copurified Nop5, which is a part of an rRNA methylating ribonucleoprotein complex, with the exosome of Sulfolobus solfataricus grown to a late stationary phase. We demonstrated the capability of Nop5 to bind to the exosome with a homotrimeric Rrp4-cap and to increase the proportion of polyadenylated RNAin vitro, suggesting that Nop5 is a dual-function protein. Since tailing of RNA probably serves to enhance RNA degradation, association of Nop5 with the archaeal exosome in the stationary phase may enhance tailing and degradation of RNA as survival strategy.

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Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Cited by 120 publications

References 36 publications

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity

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

Nop5 interacts with the archaeal RNA exosome

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