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18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences.

Fayet-Lebaron E, Atzorn V, Henry Y, Kiss T - EMBO J. (2009)

Bottom Line: Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3'-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA.The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs.We also demonstrate that besides the 18S recognition motifs, the distal part of the 3'-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, IFR109 CNRS, Université Paul Sabatier, Toulouse, France.

ABSTRACT
The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of 'orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 'orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3'-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3'-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

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Effects of altered ACA box positioning on 18S processing. (A) Structure and expression of mutant snR30 snoRNAs. The 3′-terminal portion of yeast snR30 is shown. The conserved m1, m2 and ACA motifs (boxed), deleted (underlined) and inserted (G) nucleotides are indicated. The pR30, pR30i1, pR30d1 and pR30d2 expression plasmids were transformed into the GAL∷snR30 strain and expression of the mutant snR30 RNAs was monitored by northern blot analysis after grown on glucose for 24 h. Accumulation of snR30 in non-transformed (no plasmid) cells grown either on glucose or galactose was also tested. (B) Processing of yeast pre-rRNA. Accumulation of 18S and 25S rRNA as well as 35S, 23S and 20S pre-rRNAs was monitored by Northern blot analysis.
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f5: Effects of altered ACA box positioning on 18S processing. (A) Structure and expression of mutant snR30 snoRNAs. The 3′-terminal portion of yeast snR30 is shown. The conserved m1, m2 and ACA motifs (boxed), deleted (underlined) and inserted (G) nucleotides are indicated. The pR30, pR30i1, pR30d1 and pR30d2 expression plasmids were transformed into the GAL∷snR30 strain and expression of the mutant snR30 RNAs was monitored by northern blot analysis after grown on glucose for 24 h. Accumulation of snR30 in non-transformed (no plasmid) cells grown either on glucose or galactose was also tested. (B) Processing of yeast pre-rRNA. Accumulation of 18S and 25S rRNA as well as 35S, 23S and 20S pre-rRNAs was monitored by Northern blot analysis.

Mentions: Proper function of H/ACA pseudouridylation guide RNPs requires a 14- to 15-nt-long distance between the selected uridine and the H or ACA box of the guide RNA (Ganot et al, 1997a; Bortolin et al, 1999). We noticed that the m1/m2 rRNA recognition element of all vertebrate, yeast and protozoan snR30/U17 snoRNAs is located invariantly seven nucleotides upstream of the ACA box (Figure 3A). To test the functional significance of this structural conservation of snR30/U17 snoRNAs, mutant snR30 RNAs with increased (R30i1) or decreased (R30d1 and R30d2) ACA-m1/m2 spacing were expressed in the GAL∷snR30 strain (Figure 5A). Northern blot analysis was used to confirm accumulation of the mutant snR30 RNAs and to monitor 18S processing in the transformed cells after shifting to glucose-containing medium (Figure 5A and B). Insertion of one nucleotide between the ACA box and 3′-terminal stem of the R30i1 snoRNA had no detectable effect on rRNA processing (Figure 5B, lane 6). In contrast, removal of the A601 residue upstream of the ACA box of R30d1 had no significant effect on 18S accumulation, but reduced the steady-state level of the 20S pre-rRNA and increased accumulation of the 35S and 23S pre-rRNAs, indicating that rRNA processing was slightly compromised in these cells (lane 4). Indeed, placing the ACA box two nucleotides closer to the m1/m2 element of R30d2 by removal of the A601 and G602 tail residues almost fully abolished 20S and 18S accumulation (lane 5), demonstrating that correct spacing of the 18S recognition element and the ACA box of snR30 is essential for efficient pre-rRNA processing.


18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote-specific 18S sequences.

Fayet-Lebaron E, Atzorn V, Henry Y, Kiss T - EMBO J. (2009)

Effects of altered ACA box positioning on 18S processing. (A) Structure and expression of mutant snR30 snoRNAs. The 3′-terminal portion of yeast snR30 is shown. The conserved m1, m2 and ACA motifs (boxed), deleted (underlined) and inserted (G) nucleotides are indicated. The pR30, pR30i1, pR30d1 and pR30d2 expression plasmids were transformed into the GAL∷snR30 strain and expression of the mutant snR30 RNAs was monitored by northern blot analysis after grown on glucose for 24 h. Accumulation of snR30 in non-transformed (no plasmid) cells grown either on glucose or galactose was also tested. (B) Processing of yeast pre-rRNA. Accumulation of 18S and 25S rRNA as well as 35S, 23S and 20S pre-rRNAs was monitored by Northern blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664660&req=5

f5: Effects of altered ACA box positioning on 18S processing. (A) Structure and expression of mutant snR30 snoRNAs. The 3′-terminal portion of yeast snR30 is shown. The conserved m1, m2 and ACA motifs (boxed), deleted (underlined) and inserted (G) nucleotides are indicated. The pR30, pR30i1, pR30d1 and pR30d2 expression plasmids were transformed into the GAL∷snR30 strain and expression of the mutant snR30 RNAs was monitored by northern blot analysis after grown on glucose for 24 h. Accumulation of snR30 in non-transformed (no plasmid) cells grown either on glucose or galactose was also tested. (B) Processing of yeast pre-rRNA. Accumulation of 18S and 25S rRNA as well as 35S, 23S and 20S pre-rRNAs was monitored by Northern blot analysis.
Mentions: Proper function of H/ACA pseudouridylation guide RNPs requires a 14- to 15-nt-long distance between the selected uridine and the H or ACA box of the guide RNA (Ganot et al, 1997a; Bortolin et al, 1999). We noticed that the m1/m2 rRNA recognition element of all vertebrate, yeast and protozoan snR30/U17 snoRNAs is located invariantly seven nucleotides upstream of the ACA box (Figure 3A). To test the functional significance of this structural conservation of snR30/U17 snoRNAs, mutant snR30 RNAs with increased (R30i1) or decreased (R30d1 and R30d2) ACA-m1/m2 spacing were expressed in the GAL∷snR30 strain (Figure 5A). Northern blot analysis was used to confirm accumulation of the mutant snR30 RNAs and to monitor 18S processing in the transformed cells after shifting to glucose-containing medium (Figure 5A and B). Insertion of one nucleotide between the ACA box and 3′-terminal stem of the R30i1 snoRNA had no detectable effect on rRNA processing (Figure 5B, lane 6). In contrast, removal of the A601 residue upstream of the ACA box of R30d1 had no significant effect on 18S accumulation, but reduced the steady-state level of the 20S pre-rRNA and increased accumulation of the 35S and 23S pre-rRNAs, indicating that rRNA processing was slightly compromised in these cells (lane 4). Indeed, placing the ACA box two nucleotides closer to the m1/m2 element of R30d2 by removal of the A601 and G602 tail residues almost fully abolished 20S and 18S accumulation (lane 5), demonstrating that correct spacing of the 18S recognition element and the ACA box of snR30 is essential for efficient pre-rRNA processing.

Bottom Line: Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3'-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA.The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs.We also demonstrate that besides the 18S recognition motifs, the distal part of the 3'-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, IFR109 CNRS, Université Paul Sabatier, Toulouse, France.

ABSTRACT
The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of 'orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 'orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3'-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3'-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

Show MeSH
Related in: MedlinePlus