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

Abstract

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...

C/D‐box snoRNAs form methylating and non‐methylating ribonucleoprotein complexes: Old dogs show new tricks

“…The antisense boxes recognize sequences in target RNAs resulting in the fifth nucleotide upstream of the D or D′ box being 2′-O-methylated by the methylase fibrillarin [1], which is properly positioned in the complex. The antisense boxes in eukaryotes vary in length (10–21 nt), but only a maximum of 10 nt can be used for modifications, due to the structure of the snoRNP [23]. With a few exceptions, such as the targeted U6 snRNA [24], SNORDs modify ribosomal RNAs at around 100 sites.…”

The multistructural forms of box C/D ribonucleoprotein particles

“…Several RNA-free Nop5-fibrillarin structures showed an even greater range of NTD placement through rotation around the hinge N (Ye et al 2009). Interestingly, when Ss box C/D RNP was crystallized using a symmetric box C/D RNA and the corresponding target RNA, NTD and the bound fibrillarin was observed to be placed perfectly for catalyzing methylation of the target nucleo-tide (Lin et al 2011;Yang et al 2016). This conformation of NTD is believed to represent one competent for catalysis.…”

“…By regulating the box C/D RNA stem or by changing Nop56/58 conformation, each snoRNP may form its function-specific assembly. For instance, though the work of Yang and coworkers elegantly demonstrated that the in vitro assembled Ss mono-box C/D RNP contains a tight and narrow target-binding channel allowing for a constant pairing of 10-nt duplex (Yang et al 2016), the work by van Nues and Watkins showed that the C ′ /D ′ motifs in yeast snR13, snR48, and U18 could guide methylation of two nucleotide positions of the target RNA, suggesting a structural plasticity in snoRNPs especially around the C ′ /D ′ motif (van Nues and Watkins 2017). However, it remains to be shown experimentally how these snoRNPs accommodate the plasticity.…”

Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function

“… 40 Interestingly, recent structural analysis of archaeal sRNPs indicate that only 10 basepaired nucleotides can be accommodated during catalysis, leading to the suggestion that the additional basepairing interactions facilitate accurate and efficient snoRNP recruitment and that the subsequent unwinding, which is necessary for modification, extends the residence time of the snoRNA on the pre-rRNA to prevent premature or mis-folding of the target site. 41 …”