Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2'-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs

In eukaryotic cells, the site-specific 2′-O-ribose methy-lation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2′-O-methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10–21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2′-O-methylation guide snoRNAs. However, in contrast to all of the known 2′-O-methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2′-O-methy-lated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2′-O-methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.     

Mining small RNA sequencing data: a new approach to identify small nucleolar RNAs in Arabidopsis

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that direct 2′-O-methylation or pseudouridylation on ribosomal RNAs or spliceosomal small nuclear RNAs. These modifications are needed to modulate the activity of ribosomes and spliceosomes. A comprehensive repertoire of snoRNAs is needed to expand the knowledge of these modifications. The sequences corresponding to snoRNAs in 18–26-nt small RNA sequencing data have been rarely explored and remain as a hidden treasure for snoRNA annotation. Here, we showed the enrichment of small RNAs at Arabidopsis snoRNA termini and developed a computational approach to identify snoRNAs on the basis of this characteristic. The approach successfully uncovered the full-length sequences of 144 known Arabidopsis snoRNA genes, including some snoRNAs with improved 5′- or 3′-end annotation. In addition, we identified 27 and 17 candidates for novel box C/D and box H/ACA snoRNAs, respectively. Northern blot analysis and sequencing data from parallel analysis of RNA ends confirmed the expression and the termini of the newly predicted snoRNAs. Our study especially expanded on the current knowledge of box H/ACA snoRNAs and snoRNA species targeting snRNAs. In this study, we demonstrated that the use of small RNA sequencing data can increase the complexity and the accuracy of snoRNA annotation.     

Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae

Methylation of ribose sugars at the 2′-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2′-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5′ central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D′ box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications.     

The Putative RNA Helicase Dbp4p Is Required for Release of the U14 snoRNA from Preribosomes in Saccharomyces cerevisiae

Around 70 yeast snoRNAs guide rRNA modification, frequently forming base-paired interactions predicted to be very stable at physiological temperatures. Eighteen putative RNA helicases are required for ribosome synthesis, but their actual substrates were not known. We report that depletion of the DEAD box helicase Dbp4p dramatically increased cosedimentation of the snoRNAs U14 and snR41 with preribosomes. Cosedimentation was maintained after deproteinization by proteinase K, indicating that the snoRNAs remained base paired to the pre-rRNA. Affinity purification showed that U14 was strongly accumulated in early 90S preribosomes and depleted from later pre-40S complexes. U14 is required for pre-rRNA processing, and depletion of Dbp4p caused a very similar pre-rRNA processing defect, perhaps due to the reduced pool of free U14. Point mutations in helicase motifs I and III of Dbp4p blocked release of U14 from preribosomes. We conclude that the helicase activity of Dbp4p is required to unwind U14 and snR41 from the pre-rRNA.     

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2′-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams–Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3′-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3′-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N⁷-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.     

Profiling of RNA ribose methylation in Arabidopsis thaliana

Eukaryotic rRNAs and snRNAs are decorated with abundant 2′-O-methylated nucleotides (Nm) that are predominantly synthesized by box C/D snoRNA-guided enzymes. In the model plant Arabidopsis thaliana, C/D snoRNAs have been well categorized, but there is a lack of systematic mapping of Nm. Here, we applied RiboMeth-seq to profile Nm in cytoplasmic, chloroplast and mitochondrial rRNAs and snRNAs. We identified 111 Nm in cytoplasmic rRNAs and 19 Nm in snRNAs and assigned guide for majority of the detected sites using an updated snoRNA list. At least four sites are directed by guides with multiple specificities as shown in yeast. We found that C/D snoRNAs frequently form extra pairs with nearby sequences of methylation sites, potentially facilitating the substrate binding. Chloroplast and mitochondrial rRNAs contain five almost identical methylation sites, including two novel sites mediating ribosomal subunit joining. Deletion of FIB1 or FIB2 gene reduced the accumulation of C/D snoRNA and rRNA methylation with FIB1 playing a bigger role in methylation. Our data reveal the comprehensive 2′-O-methylation maps for Arabidopsis rRNAs and snRNAs and would facilitate study of their function and biosynthesis.     

Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase

In yeast, three small nucleolar RNAs (snoRNAs) are essential for the processing of pre-ribosomal RNA—U3, U14 and snR30—whereas 72 non-essential snoRNAs direct site-specific modification of pre-rRNA. We applied a quantitative screen for alterations in the pre-ribosome association to all 75 yeast snoRNAs in strains depleted of eight putative helicases implicated in 40S subunit synthesis. For the modification-guide snoRNAs, we found no clear evidence for the involvement of these helicases in the association or dissociation of pre-ribosomes. However, the DEAD box helicase Rok1 was required specifically for the release of snR30. Point mutations in motif I, but not in motif III, of the helicase domain of Rok1 impaired the release of snR30, but this was less marked than in strains depleted of Rok1, and resulted in a dominant-negative growth phenotype. Dissociation of U3 and U14 from pre-ribosomes is also dependent on helicases, suggesting that release of the essential snoRNAs might differ mechanistically from release of the modification-guide snoRNAs.     

Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.     

Identification and functional implications of pseudouridine RNA modification on small noncoding RNAs in the mammalian pathogen Trypanosoma brucei

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2′-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts.     

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.     

The bipartite architecture of the sRNA in an archaeal box C/D complex is a primary determinant of specificity

The archaeal box C/D sRNP, the enzyme responsible for 2′-O-methylation of rRNA and tRNA, possesses a nearly perfect axis of symmetry and bipartite structure. This RNP contains two platforms for the assembly of protein factors, the C/D and C′/D′ motifs, acting in conjunction with two guide sequences to direct methylation of a specific 2′-hydroxyl group in a target RNA. While this suggests that a functional asymmetric single-site complex complete with guide sequence and a single box C/D motif should be possible, previous work has demonstrated such constructs are not viable. To understand the basis for a bipartite RNP, we have designed and assayed the activity and specificity of a series of synthetic RNPs that represent a systematic reduction of the wild-type RNP to a fully single-site enzyme. This reduced RNP is active and exhibits all of the characteristics of wild-type box C/D RNPs except it is nonspecific with respect to the site of 2′-O-methylation. Our results demonstrate that protein–protein crosstalk through Nop5p dimerization is not required, but that architecture plays a crucial role in directing methylation activity with both C/D and C′/D′ motifs being required for specificity.     

Yeast Rrp8p,a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m¹A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m¹A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.     

Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy

Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2′-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains—the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.     

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3′-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3′–hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3′-hinge was more important than the left. Increasing the length of the U3 hinge–ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5′- and 3′-hinge interactions with the ETS or by shifting the base pairing of the U3 3′-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3′-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing.     

Strong dependence between functional domains in a dual-function snoRNA infers coupling of rRNA processing and modification events

Most small nucleolar RNAs (snoRNAs) guide rRNA nucleotide modifications, some participate in pre-rRNA cleavages, and a few have both functions. These activities involve direct base-pairing of the snoRNA with pre-rRNA using different domains. It is not known if the modification and processing functions occur independently or in a coordinated manner. We address this question by mutational analysis of a yeast box H/ACA snoRNA that mediates both processing and modification. This snoRNA (snR10) contains canonical 5′- and 3′-hairpin structures with a guide domain for pseudouridylation in the 3′ hairpin. Our functional mapping results show that: (i) processing requires the 5′ hairpin exclusively, in particular a 7-nt element; (ii) loss of the 3′ hairpin or pseudouridine does not affect rRNA processing; (iii) a single nucleotide insertion in the guide domain shifts modification to an adjacent uridine in rRNA, and severely impairs both processing and cell growth; and (iv) the deleterious effects of the insertion mutation depend on the presence of the processing element in the 5′ hairpin, but not modification of the novel site. Together, the results suggest that the snoRNA hairpins function in a coordinated manner and that their interactions with pre-rRNA could be coupled.     

Box C/D snoRNP catalysed methylation is aided by additional pre-rRNA base-pairing

2'-O-methylation of eukaryotic ribosomal RNA (r)RNA, essential for ribosome function, is catalysed by box C/D small nucleolar (sno)RNPs. The RNA components of these complexes (snoRNAs) contain one or two guide sequences, which, through base-pairing, select the rRNA modification site. Adjacent to the guide sequences are protein-binding sites (the C/D or C'/D' motifs). Analysis of >2000 yeast box C/D snoRNAs identified additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site. This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold. Sequence analysis, combined with RNA-protein crosslinking in Saccharomyces cerevisiae, identified highly divergent box C'/D' motifs that are bound by snoRNP proteins. In vivo rRNA methylation assays showed these to be active. Our data suggest roles for non-catalytic subunits (Nop56 and Nop58) in rRNA binding and support an asymmetric model for box C/D snoRNP organization. The study provides novel insights into the extent of the snoRNA-rRNA interactions required for efficient methylation and the structural organization of the snoRNPs.     

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Eukaryotic nucleoli contain a large family of box C+D small nucleolar RNA (snoRNA) species, all of which are associated with a common protein Nop1p/fibrillarin. Nop58p was identified in a screen for synthetic lethality with Nop1p and shown to be an essential nucleolar protein. Here we report that a Protein A-tagged version of Nop58p coprecipitates all tested box C+D snoRNAs and that genetic depletion of Nop58p leads to the loss of all tested box C+D snoRNAs. The box H+ACA class of snoRNAs are not coprecipitated with Nop58p, and are not codepleted. The yeast box C+D snoRNAs include two species, U3 and U14, that are required for the early cleavages in pre-rRNA processing. Consistent with this, Nop58p depletion leads to a strong inhibition of pre-rRNA processing and 18S rRNA synthesis. Unexpectedly, depletion of Nop58p leads to the accumulation of 3' extended forms of U3 and U24, showing that the protein is also involved in snoRNA synthesis. Nop58p is the second common component of the box C+D snoRNPs to be identified and the first to be shown to be required for the stability and for the synthesis of these snoRNAs.     

Box C/D sRNA stem ends act as stabilizing anchors for box C/D di-sRNPs

Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′-O-methylation, one rRNA modification type in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs have demonstrated the dimeric sRNP (di-sRNP) architecture, which has been corroborated by nuclear magnetic resonance (NMR) studies. Due to limitations of the structural techniques, the orientation of the box C/D sRNAs has remained unclear. Here, we have used cryo-EM to elucidate the sRNA orientation in a M. jannaschii box C/D di-sRNP. The cryo-EM reconstruction suggests a parallel orientation of the two sRNAs. Biochemical and structural analyses of sRNPs assembled with mutant sRNAs indicate a potential interaction between the sRNA stem ends. Our results suggest that the parallel arrangement of the sRNAs juxtaposes their stem ends into close proximity to allow for a stabilizing interaction that helps maintain the di-sRNP architecture.