Conserved spacing between the box C/D and C′/D′ RNPs of the archaeal box C/D sRNP complex is required for efficient 2′-O-methylation of target RNAs

Tran, Elizabeth; Zhang, Xinxin; Lackey, Lela; Maxwell, Elizabeth S.

doi:10.1261/rna.7223405

Cited by 42 publications

(51 citation statements)

References 43 publications

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“…Analysis of this large dataset showed that 57.8% of the spacers are 12 nt long (Fig. 4A), confirming a previous report (32). Only 14.2% of spacers are <12 nt long; however, these short spacers have significantly lower percentages (20.1-36%) of predicted rRNA targets compared with the spacers of ≥12 nt (46.7-70%).…”

Section: Discussionsupporting

confidence: 88%

“…The data show that extensive guide-substrate pairing (12 bp in this case) inhibits the modification in a temperature-dependent manner. An archaeal C/D RNA with spacers shorter or longer than 12 nt was previously shown to be defective in guiding modification (32). We confirmed that short spacers are not functional, but did not find that long spacers (up to 15 nt) are defective.…”

Section: Resultssupporting

confidence: 71%

“…Archaeal C/D RNAs have a compact structure, with the spacer region length constrained to 12 nt (32). Recently, 489 C/D RNAs were compiled from the RNA-Seq data from seven archaea, and 719 guide-rRNA interactions were predicted (39).…”

Section: Discussionmentioning

confidence: 99%

“…The spacer sequence located between boxes D and C′ and boxes D′ and C is of variable length in C/D RNAs. In archaeal C/D RNAs, the spacer is most frequently 12 nt long (32); however, in eukaryotes, spacer length varies widely and can potentially form 10-21 bp with the substrate (5,32,33). This raises an important question of how C/D RNPs can accommodate differently sized spacers and guide-substrate duplexes while still maintaining the specificity of modification.…”

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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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Section: Discussionsupporting

confidence: 88%

Section: Resultssupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

mentioning

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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“…Optimal methylation activity requires that both box C/D and C′/D′ RNPs be juxtaposed and spatially positioned within the same sRNP complex. 30,31,35 Incomplete assembly caused by mutation, loss of protein components at either the C/D or C′/D′ motif, or alterations in inter-RNP spacing, results in lost or reduced methylation activity from both RNPs. Unfortunately, a complete understanding of box C/D RNP assembly is presently lacking and the nature of inter-RNP interactions is still undefined.…”

Section: Introductionmentioning

confidence: 99%