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

van Nues RW, Granneman S, Kudla G, Sloan KE, Chicken M, Tollervey D, Watkins NJ - EMBO J. (2011)

Bottom Line: This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.

ABSTRACT
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.

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Related in: MedlinePlus

Sequence alignments of box C/D snoRNAs. (A) Homologues for each of the S. cerevisiae box C/D snoRNAs were retrieved from the fungal genomic sequence databases and aligned. Two example alignments, using a limited subset of the sequences for snR74 and snR75, are shown. The sequence is shown 5′–3′ and the position of the box sequences are indicated, with the consensus sequence shown at the bottom. The rRNA target (3′–5′) is shown in white on a red background. The extra base-pairing target of snR75 is shown in white with a blue background. Identical sequences: white with a black background; conserved sequences: black with a grey background. Brackets indicate possible intra-molecular base-pairing. Scer: Saccharomyces cerevisiae; Cgla: Candida glabrata; Klac: Kluyveromyces lactis; Lelo: Lodderomyces elongisporus; Wano: Wickerhamomyces anomalus (Pichia anomala); Sjap: Schizosaccharomyces japonicas; Tree: Trichoderma reesei (Hypocrea jecorina); Tsti: Talaromyces stipitatus; Acla: Aspergillus clavatus; Nfis: Neosartorya fischeri; Cpos: Coccidioides posadasii; Pans: Podospora anserine. (B) The D′ and C′ sequences of the S. cerevisiae box C/D snoRNAs are shown. Insertions in the C′ boxes are indicated in red. The snoRNAs containing box C′/D′ motifs that do not appear to direct methylation are indicated in grey. (C) A schematic representation of the conservation of the sequences of the C, D, C′ and D′ boxes of the S. cerevisiae box C/D. The diagram was prepared using the WebLogo software (Crooks et al, 2004).
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f1: Sequence alignments of box C/D snoRNAs. (A) Homologues for each of the S. cerevisiae box C/D snoRNAs were retrieved from the fungal genomic sequence databases and aligned. Two example alignments, using a limited subset of the sequences for snR74 and snR75, are shown. The sequence is shown 5′–3′ and the position of the box sequences are indicated, with the consensus sequence shown at the bottom. The rRNA target (3′–5′) is shown in white on a red background. The extra base-pairing target of snR75 is shown in white with a blue background. Identical sequences: white with a black background; conserved sequences: black with a grey background. Brackets indicate possible intra-molecular base-pairing. Scer: Saccharomyces cerevisiae; Cgla: Candida glabrata; Klac: Kluyveromyces lactis; Lelo: Lodderomyces elongisporus; Wano: Wickerhamomyces anomalus (Pichia anomala); Sjap: Schizosaccharomyces japonicas; Tree: Trichoderma reesei (Hypocrea jecorina); Tsti: Talaromyces stipitatus; Acla: Aspergillus clavatus; Nfis: Neosartorya fischeri; Cpos: Coccidioides posadasii; Pans: Podospora anserine. (B) The D′ and C′ sequences of the S. cerevisiae box C/D snoRNAs are shown. Insertions in the C′ boxes are indicated in red. The snoRNAs containing box C′/D′ motifs that do not appear to direct methylation are indicated in grey. (C) A schematic representation of the conservation of the sequences of the C, D, C′ and D′ boxes of the S. cerevisiae box C/D. The diagram was prepared using the WebLogo software (Crooks et al, 2004).

Mentions: It has been proposed that snoRNA C′/D′ and C/D motifs are based on the same core consensus sequences, even though C′/D′ elements are generally less well conserved (Kiss-Laszlo et al, 1998). Surprisingly, analysis of the primary sequence failed to clearly identify C′ and/or D′ boxes in many S. cerevisiae box C/D snoRNAs. We, therefore, compared the sequence of each S. cerevisiae box C/D snoRNA across multiple yeast species (Figure 1A; Supplementary Figure S1). This enabled the identification of C′/D′ motifs in all snoRNAs (Figure 1B; Supplementary Figure S1). There was a surprising amount of variation in C′/D′ sequences between individual snoRNAs; ranging from poorly (e.g., snR51) to highly conserved (e.g., snR53) motifs. Some snoRNAs harbour sequences that are highly conserved in yeast evolution but quite distinct from the accepted consensus, for both D′ (e.g., snR73, snR70, snR51 and snR87) and C′ (e.g., snR68 and U24). Surprisingly, nine C′ boxes in S. cerevisiae aligned better to the consensus with between one or two nucleotide insertions (Figure 1B; e.g., snR50 and snR69). In some cases, insertions were present in a subset of the orthologues of a single snoRNA (e.g., snR71) and in some snoRNAs there could be up to nine nucleotides inserted in the split C′ boxes (Supplementary Figure S1; snR190 and snR76). One or two nucleotide insertions were apparent in C′ boxes from vertebrate snoRNAs (e.g., rodent U15B, HBII-234, HBII-82 and mgh28S-2411; data not shown), indicating that this is not specific to yeast. C′/D′ motifs were also highly conserved in snoRNAs that do not appear to use this motif to direct methylation (Figure 1B); the ‘guide' region adjacent to the D′ motif in each of these RNAs was not conserved and no target has been identified (Supplementary Figure S1). Indeed, some of these ‘inactive' motifs show better sequence conservation than active motifs, suggesting that the C′/D′ motif has a key role in the overall architecture of the snoRNP. The identification of many unusual C′/D′ motif sequences raised questions about the validity of the original consensus sequence. Re-evaluation of the C′ and D′ sequences confirmed that, while the original consensus sequence was correctly identified, significant divergence is tolerated in this motif (Figure 1C). The alignments also identified highly conserved regions present in several snoRNAs (e.g., snR75 and snR70; Figure 1A; Supplementary Figure S1), which do not correspond to either the box or guide regions. We speculated that these could assist in snoRNA function through providing a protein-binding site or additional base-pairing potential (see below).


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

van Nues RW, Granneman S, Kudla G, Sloan KE, Chicken M, Tollervey D, Watkins NJ - EMBO J. (2011)

Sequence alignments of box C/D snoRNAs. (A) Homologues for each of the S. cerevisiae box C/D snoRNAs were retrieved from the fungal genomic sequence databases and aligned. Two example alignments, using a limited subset of the sequences for snR74 and snR75, are shown. The sequence is shown 5′–3′ and the position of the box sequences are indicated, with the consensus sequence shown at the bottom. The rRNA target (3′–5′) is shown in white on a red background. The extra base-pairing target of snR75 is shown in white with a blue background. Identical sequences: white with a black background; conserved sequences: black with a grey background. Brackets indicate possible intra-molecular base-pairing. Scer: Saccharomyces cerevisiae; Cgla: Candida glabrata; Klac: Kluyveromyces lactis; Lelo: Lodderomyces elongisporus; Wano: Wickerhamomyces anomalus (Pichia anomala); Sjap: Schizosaccharomyces japonicas; Tree: Trichoderma reesei (Hypocrea jecorina); Tsti: Talaromyces stipitatus; Acla: Aspergillus clavatus; Nfis: Neosartorya fischeri; Cpos: Coccidioides posadasii; Pans: Podospora anserine. (B) The D′ and C′ sequences of the S. cerevisiae box C/D snoRNAs are shown. Insertions in the C′ boxes are indicated in red. The snoRNAs containing box C′/D′ motifs that do not appear to direct methylation are indicated in grey. (C) A schematic representation of the conservation of the sequences of the C, D, C′ and D′ boxes of the S. cerevisiae box C/D. The diagram was prepared using the WebLogo software (Crooks et al, 2004).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3116282&req=5

f1: Sequence alignments of box C/D snoRNAs. (A) Homologues for each of the S. cerevisiae box C/D snoRNAs were retrieved from the fungal genomic sequence databases and aligned. Two example alignments, using a limited subset of the sequences for snR74 and snR75, are shown. The sequence is shown 5′–3′ and the position of the box sequences are indicated, with the consensus sequence shown at the bottom. The rRNA target (3′–5′) is shown in white on a red background. The extra base-pairing target of snR75 is shown in white with a blue background. Identical sequences: white with a black background; conserved sequences: black with a grey background. Brackets indicate possible intra-molecular base-pairing. Scer: Saccharomyces cerevisiae; Cgla: Candida glabrata; Klac: Kluyveromyces lactis; Lelo: Lodderomyces elongisporus; Wano: Wickerhamomyces anomalus (Pichia anomala); Sjap: Schizosaccharomyces japonicas; Tree: Trichoderma reesei (Hypocrea jecorina); Tsti: Talaromyces stipitatus; Acla: Aspergillus clavatus; Nfis: Neosartorya fischeri; Cpos: Coccidioides posadasii; Pans: Podospora anserine. (B) The D′ and C′ sequences of the S. cerevisiae box C/D snoRNAs are shown. Insertions in the C′ boxes are indicated in red. The snoRNAs containing box C′/D′ motifs that do not appear to direct methylation are indicated in grey. (C) A schematic representation of the conservation of the sequences of the C, D, C′ and D′ boxes of the S. cerevisiae box C/D. The diagram was prepared using the WebLogo software (Crooks et al, 2004).
Mentions: It has been proposed that snoRNA C′/D′ and C/D motifs are based on the same core consensus sequences, even though C′/D′ elements are generally less well conserved (Kiss-Laszlo et al, 1998). Surprisingly, analysis of the primary sequence failed to clearly identify C′ and/or D′ boxes in many S. cerevisiae box C/D snoRNAs. We, therefore, compared the sequence of each S. cerevisiae box C/D snoRNA across multiple yeast species (Figure 1A; Supplementary Figure S1). This enabled the identification of C′/D′ motifs in all snoRNAs (Figure 1B; Supplementary Figure S1). There was a surprising amount of variation in C′/D′ sequences between individual snoRNAs; ranging from poorly (e.g., snR51) to highly conserved (e.g., snR53) motifs. Some snoRNAs harbour sequences that are highly conserved in yeast evolution but quite distinct from the accepted consensus, for both D′ (e.g., snR73, snR70, snR51 and snR87) and C′ (e.g., snR68 and U24). Surprisingly, nine C′ boxes in S. cerevisiae aligned better to the consensus with between one or two nucleotide insertions (Figure 1B; e.g., snR50 and snR69). In some cases, insertions were present in a subset of the orthologues of a single snoRNA (e.g., snR71) and in some snoRNAs there could be up to nine nucleotides inserted in the split C′ boxes (Supplementary Figure S1; snR190 and snR76). One or two nucleotide insertions were apparent in C′ boxes from vertebrate snoRNAs (e.g., rodent U15B, HBII-234, HBII-82 and mgh28S-2411; data not shown), indicating that this is not specific to yeast. C′/D′ motifs were also highly conserved in snoRNAs that do not appear to use this motif to direct methylation (Figure 1B); the ‘guide' region adjacent to the D′ motif in each of these RNAs was not conserved and no target has been identified (Supplementary Figure S1). Indeed, some of these ‘inactive' motifs show better sequence conservation than active motifs, suggesting that the C′/D′ motif has a key role in the overall architecture of the snoRNP. The identification of many unusual C′/D′ motif sequences raised questions about the validity of the original consensus sequence. Re-evaluation of the C′ and D′ sequences confirmed that, while the original consensus sequence was correctly identified, significant divergence is tolerated in this motif (Figure 1C). The alignments also identified highly conserved regions present in several snoRNAs (e.g., snR75 and snR70; Figure 1A; Supplementary Figure S1), which do not correspond to either the box or guide regions. We speculated that these could assist in snoRNA function through providing a protein-binding site or additional base-pairing potential (see below).

Bottom Line: This 'extra base-pairing' was also found in many human box C/D snoRNAs and can stimulate methylation by up to five-fold.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus