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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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Methylation activity of C′/D′ motifs. (A) Schematic representation of the galactose-inducible snoRNA expression cassette. The positions of the GAL promoter (GALp), ADH terminator sequence (ADHt) and exons 1 and 2 of the actin gene (E1 and E2) are shown. The positions of the Nhe I and Mlu I restriction sites, used in the cloning of the various C′/D′ fragments, are indicated. The C′/D′ sequences cloned into this cassette are shown in Supplementary Figure S10. (B, C) snoRNAs containing wild-type and mutant C′ boxes (as indicated above each lane) were transformed into yeast cells. RNA was extracted from the cells and analysed by primer extension, using primer Map1316 (upper panel), to detect rRNA methylation, and by northern hybridization (Supplementary Figure S2) to detect the expression of the snoRNA. The position of the stop corresponding to methylation of the target nucleotide, S1316 in the 18S rRNA, is indicated on the right. The snoRNA containing the C′/D′ motif from hU24 was used in all experiments to enable the comparison of the relative methylation activity of the various C′/D′ motifs.
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f3: Methylation activity of C′/D′ motifs. (A) Schematic representation of the galactose-inducible snoRNA expression cassette. The positions of the GAL promoter (GALp), ADH terminator sequence (ADHt) and exons 1 and 2 of the actin gene (E1 and E2) are shown. The positions of the Nhe I and Mlu I restriction sites, used in the cloning of the various C′/D′ fragments, are indicated. The C′/D′ sequences cloned into this cassette are shown in Supplementary Figure S10. (B, C) snoRNAs containing wild-type and mutant C′ boxes (as indicated above each lane) were transformed into yeast cells. RNA was extracted from the cells and analysed by primer extension, using primer Map1316 (upper panel), to detect rRNA methylation, and by northern hybridization (Supplementary Figure S2) to detect the expression of the snoRNA. The position of the stop corresponding to methylation of the target nucleotide, S1316 in the 18S rRNA, is indicated on the right. The snoRNA containing the C′/D′ motif from hU24 was used in all experiments to enable the comparison of the relative methylation activity of the various C′/D′ motifs.

Mentions: Since many C′/D′ motifs diverge from the consensus, we next compared the activity of several such motifs in directing rRNA methylation. To perform this, we developed an expression system for an artificial snoRNA designed to target methylation of nucleotide S1316 in the 18S rRNA (Figure 3A) and expressed under the control of a GAL promoter. This site is not naturally methylated but its modification does not affect growth (Decatur and Fournier, personal communication; data not shown). We used the human U24 C′/D′ sequence as a standard in the artificial snoRNA as this motif matches the consensus sequence and was previously used to characterize C′/D′ sequence function in yeast (Kiss-Laszlo et al, 1998; Qu et al, 2011). Several C′/D′ motifs were cloned into this snoRNA construct, including the divergent motifs from snR47, snR51 and snR70, the ‘split' motif from snR78 and ‘inactive' C′/D′ motifs from snR39, snR50, snR55 and snR57, that are not naturally adjacent to a guide sequence. The resulting plasmids were transformed into S. cerevisiae grown on galactose medium to induce snoRNA expression, and RNA was extracted. Methylation status was determined by primer extension (Figure 3B; Figure 5B (snR70)) and snoRNA levels were monitored by northern blotting (Supplementary Figure S2).


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)

Methylation activity of C′/D′ motifs. (A) Schematic representation of the galactose-inducible snoRNA expression cassette. The positions of the GAL promoter (GALp), ADH terminator sequence (ADHt) and exons 1 and 2 of the actin gene (E1 and E2) are shown. The positions of the Nhe I and Mlu I restriction sites, used in the cloning of the various C′/D′ fragments, are indicated. The C′/D′ sequences cloned into this cassette are shown in Supplementary Figure S10. (B, C) snoRNAs containing wild-type and mutant C′ boxes (as indicated above each lane) were transformed into yeast cells. RNA was extracted from the cells and analysed by primer extension, using primer Map1316 (upper panel), to detect rRNA methylation, and by northern hybridization (Supplementary Figure S2) to detect the expression of the snoRNA. The position of the stop corresponding to methylation of the target nucleotide, S1316 in the 18S rRNA, is indicated on the right. The snoRNA containing the C′/D′ motif from hU24 was used in all experiments to enable the comparison of the relative methylation activity of the various C′/D′ motifs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Methylation activity of C′/D′ motifs. (A) Schematic representation of the galactose-inducible snoRNA expression cassette. The positions of the GAL promoter (GALp), ADH terminator sequence (ADHt) and exons 1 and 2 of the actin gene (E1 and E2) are shown. The positions of the Nhe I and Mlu I restriction sites, used in the cloning of the various C′/D′ fragments, are indicated. The C′/D′ sequences cloned into this cassette are shown in Supplementary Figure S10. (B, C) snoRNAs containing wild-type and mutant C′ boxes (as indicated above each lane) were transformed into yeast cells. RNA was extracted from the cells and analysed by primer extension, using primer Map1316 (upper panel), to detect rRNA methylation, and by northern hybridization (Supplementary Figure S2) to detect the expression of the snoRNA. The position of the stop corresponding to methylation of the target nucleotide, S1316 in the 18S rRNA, is indicated on the right. The snoRNA containing the C′/D′ motif from hU24 was used in all experiments to enable the comparison of the relative methylation activity of the various C′/D′ motifs.
Mentions: Since many C′/D′ motifs diverge from the consensus, we next compared the activity of several such motifs in directing rRNA methylation. To perform this, we developed an expression system for an artificial snoRNA designed to target methylation of nucleotide S1316 in the 18S rRNA (Figure 3A) and expressed under the control of a GAL promoter. This site is not naturally methylated but its modification does not affect growth (Decatur and Fournier, personal communication; data not shown). We used the human U24 C′/D′ sequence as a standard in the artificial snoRNA as this motif matches the consensus sequence and was previously used to characterize C′/D′ sequence function in yeast (Kiss-Laszlo et al, 1998; Qu et al, 2011). Several C′/D′ motifs were cloned into this snoRNA construct, including the divergent motifs from snR47, snR51 and snR70, the ‘split' motif from snR78 and ‘inactive' C′/D′ motifs from snR39, snR50, snR55 and snR57, that are not naturally adjacent to a guide sequence. The resulting plasmids were transformed into S. cerevisiae grown on galactose medium to induce snoRNA expression, and RNA was extracted. Methylation status was determined by primer extension (Figure 3B; Figure 5B (snR70)) and snoRNA levels were monitored by northern blotting (Supplementary Figure S2).

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