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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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Analysis of yeast strains expressing mutant 18S and snR30 RNAs. (A) Expression constructs used to express wild-type (pTH25) and mutant (pTH25rm1 and pTH25rm2) 18S rRNAs and mutant snR30 (pR30m1 and pR30m2) snoRNAs. Tag sequences in the 18S and 25S rRNA genes are indicated by open boxes (Beltrame and Tollervey, 1992). The GAL7 and SNR5 promoters (SNR5-P) and the SNR5 terminator (SNR5-T) are shown. Nucleotide alterations introduced into 18S (18Srm1 and 18Srm2) or snR30 (snR30m1 and snR30m2) RNAs are shown. The m1 and m2 motifs of snR30 are boxed. (B) Growth properties of yeast NOY504 strains not transformed (no plasmid) or transformed with the indicated expression plasmids on galactose medium at 37 and 25°C. (C) Expression of mutant 18S RNAs. RNAs extracted from NOY504 cells transformed with the indicated plasmids were separated on a 1.2% agarose-formaldehyde gel and blotted onto a nylon membrane. Accumulation of 25S and 18S rRNAs and 20S pre-rRNA was determined by probing the blots with oligonucleotide probes specific for the tag sequences in the ectopically expressed 18S and 25S rRNAs or complementary to the ITS1 region of yeast 35S pre-rRNA (20S pre-RNA). Growth temperatures are indicated. (D) Expression of mutant snR30m1 and snR30m2 snoRNAs. RNAs extracted from yeast NOY504 strains transformed with the indicated plasmids were annealed with an antisense RNA probe complementary to wild-type snR30 and digested with a mixture of RNase A and T1. The protected fragments were separated on a 6% sequencing gel. Structures and sizes of the protected probe RNAs are shown. Positions of the protected RNAs corresponding to the wild-type and the mutant snR30 RNAs are indicated on the right. Lane control, mapping performed with E. coli tRNA. Lane M, size markers. (E) Processing of 18S rRNA carrying an altered rm1 motif can be restored by compensatory base changes in snR30. The nucleotide changes in 18Srm1b and snR30m1b RNAs are shown. Yeast NOY504 strains transformed with the indicated plasmids were grown on galactose medium. Accumulation of mutant snR30 and 18S RNAs was monitored by RNase A/T1 mapping and northern blot hybridization, respectively.
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f4: Analysis of yeast strains expressing mutant 18S and snR30 RNAs. (A) Expression constructs used to express wild-type (pTH25) and mutant (pTH25rm1 and pTH25rm2) 18S rRNAs and mutant snR30 (pR30m1 and pR30m2) snoRNAs. Tag sequences in the 18S and 25S rRNA genes are indicated by open boxes (Beltrame and Tollervey, 1992). The GAL7 and SNR5 promoters (SNR5-P) and the SNR5 terminator (SNR5-T) are shown. Nucleotide alterations introduced into 18S (18Srm1 and 18Srm2) or snR30 (snR30m1 and snR30m2) RNAs are shown. The m1 and m2 motifs of snR30 are boxed. (B) Growth properties of yeast NOY504 strains not transformed (no plasmid) or transformed with the indicated expression plasmids on galactose medium at 37 and 25°C. (C) Expression of mutant 18S RNAs. RNAs extracted from NOY504 cells transformed with the indicated plasmids were separated on a 1.2% agarose-formaldehyde gel and blotted onto a nylon membrane. Accumulation of 25S and 18S rRNAs and 20S pre-rRNA was determined by probing the blots with oligonucleotide probes specific for the tag sequences in the ectopically expressed 18S and 25S rRNAs or complementary to the ITS1 region of yeast 35S pre-rRNA (20S pre-RNA). Growth temperatures are indicated. (D) Expression of mutant snR30m1 and snR30m2 snoRNAs. RNAs extracted from yeast NOY504 strains transformed with the indicated plasmids were annealed with an antisense RNA probe complementary to wild-type snR30 and digested with a mixture of RNase A and T1. The protected fragments were separated on a 6% sequencing gel. Structures and sizes of the protected probe RNAs are shown. Positions of the protected RNAs corresponding to the wild-type and the mutant snR30 RNAs are indicated on the right. Lane control, mapping performed with E. coli tRNA. Lane M, size markers. (E) Processing of 18S rRNA carrying an altered rm1 motif can be restored by compensatory base changes in snR30. The nucleotide changes in 18Srm1b and snR30m1b RNAs are shown. Yeast NOY504 strains transformed with the indicated plasmids were grown on galactose medium. Accumulation of mutant snR30 and 18S RNAs was monitored by RNase A/T1 mapping and northern blot hybridization, respectively.

Mentions: The rm1 and rm2 motifs are located within an internal domain of 18S rRNA that is specific for eukaryotic 18S rRNAs and that, at least on the primary sequence, is far from pre-rRNA-processing sites (Figure 3D). Therefore, we first investigated whether the rm1 and rm2 sequences are essential for pre-rRNA processing and 18S rRNA accumulation. We utilized the pTH25 rRNA expression construct that carries the yeast Saccharomyces cerevisiae 35S pre-rRNA gene under the control of the galactose-inducible GAL7 promoter (Beltrame and Tollervey, 1992; Henry et al, 1994) (Figure 4A). To facilitate monitoring of 18S and 25S expression from the plasmid-borne rDNA allele, neutral sequence tags had been inserted into the 5′-terminal regions of the 18S and 25S rRNA genes. Moreover, we introduced nucleotide changes into the rm1 and rm2 motifs of the 18S rRNA gene that reduced the potential of the resulting 18Srm1 (pTH25rm1) and 18Srm2 (pTH25rm2) rRNAs for base pairing with the m1 and m2 motifs of snR30, respectively.


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)

Analysis of yeast strains expressing mutant 18S and snR30 RNAs. (A) Expression constructs used to express wild-type (pTH25) and mutant (pTH25rm1 and pTH25rm2) 18S rRNAs and mutant snR30 (pR30m1 and pR30m2) snoRNAs. Tag sequences in the 18S and 25S rRNA genes are indicated by open boxes (Beltrame and Tollervey, 1992). The GAL7 and SNR5 promoters (SNR5-P) and the SNR5 terminator (SNR5-T) are shown. Nucleotide alterations introduced into 18S (18Srm1 and 18Srm2) or snR30 (snR30m1 and snR30m2) RNAs are shown. The m1 and m2 motifs of snR30 are boxed. (B) Growth properties of yeast NOY504 strains not transformed (no plasmid) or transformed with the indicated expression plasmids on galactose medium at 37 and 25°C. (C) Expression of mutant 18S RNAs. RNAs extracted from NOY504 cells transformed with the indicated plasmids were separated on a 1.2% agarose-formaldehyde gel and blotted onto a nylon membrane. Accumulation of 25S and 18S rRNAs and 20S pre-rRNA was determined by probing the blots with oligonucleotide probes specific for the tag sequences in the ectopically expressed 18S and 25S rRNAs or complementary to the ITS1 region of yeast 35S pre-rRNA (20S pre-RNA). Growth temperatures are indicated. (D) Expression of mutant snR30m1 and snR30m2 snoRNAs. RNAs extracted from yeast NOY504 strains transformed with the indicated plasmids were annealed with an antisense RNA probe complementary to wild-type snR30 and digested with a mixture of RNase A and T1. The protected fragments were separated on a 6% sequencing gel. Structures and sizes of the protected probe RNAs are shown. Positions of the protected RNAs corresponding to the wild-type and the mutant snR30 RNAs are indicated on the right. Lane control, mapping performed with E. coli tRNA. Lane M, size markers. (E) Processing of 18S rRNA carrying an altered rm1 motif can be restored by compensatory base changes in snR30. The nucleotide changes in 18Srm1b and snR30m1b RNAs are shown. Yeast NOY504 strains transformed with the indicated plasmids were grown on galactose medium. Accumulation of mutant snR30 and 18S RNAs was monitored by RNase A/T1 mapping and northern blot hybridization, respectively.
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f4: Analysis of yeast strains expressing mutant 18S and snR30 RNAs. (A) Expression constructs used to express wild-type (pTH25) and mutant (pTH25rm1 and pTH25rm2) 18S rRNAs and mutant snR30 (pR30m1 and pR30m2) snoRNAs. Tag sequences in the 18S and 25S rRNA genes are indicated by open boxes (Beltrame and Tollervey, 1992). The GAL7 and SNR5 promoters (SNR5-P) and the SNR5 terminator (SNR5-T) are shown. Nucleotide alterations introduced into 18S (18Srm1 and 18Srm2) or snR30 (snR30m1 and snR30m2) RNAs are shown. The m1 and m2 motifs of snR30 are boxed. (B) Growth properties of yeast NOY504 strains not transformed (no plasmid) or transformed with the indicated expression plasmids on galactose medium at 37 and 25°C. (C) Expression of mutant 18S RNAs. RNAs extracted from NOY504 cells transformed with the indicated plasmids were separated on a 1.2% agarose-formaldehyde gel and blotted onto a nylon membrane. Accumulation of 25S and 18S rRNAs and 20S pre-rRNA was determined by probing the blots with oligonucleotide probes specific for the tag sequences in the ectopically expressed 18S and 25S rRNAs or complementary to the ITS1 region of yeast 35S pre-rRNA (20S pre-RNA). Growth temperatures are indicated. (D) Expression of mutant snR30m1 and snR30m2 snoRNAs. RNAs extracted from yeast NOY504 strains transformed with the indicated plasmids were annealed with an antisense RNA probe complementary to wild-type snR30 and digested with a mixture of RNase A and T1. The protected fragments were separated on a 6% sequencing gel. Structures and sizes of the protected probe RNAs are shown. Positions of the protected RNAs corresponding to the wild-type and the mutant snR30 RNAs are indicated on the right. Lane control, mapping performed with E. coli tRNA. Lane M, size markers. (E) Processing of 18S rRNA carrying an altered rm1 motif can be restored by compensatory base changes in snR30. The nucleotide changes in 18Srm1b and snR30m1b RNAs are shown. Yeast NOY504 strains transformed with the indicated plasmids were grown on galactose medium. Accumulation of mutant snR30 and 18S RNAs was monitored by RNase A/T1 mapping and northern blot hybridization, respectively.
Mentions: The rm1 and rm2 motifs are located within an internal domain of 18S rRNA that is specific for eukaryotic 18S rRNAs and that, at least on the primary sequence, is far from pre-rRNA-processing sites (Figure 3D). Therefore, we first investigated whether the rm1 and rm2 sequences are essential for pre-rRNA processing and 18S rRNA accumulation. We utilized the pTH25 rRNA expression construct that carries the yeast Saccharomyces cerevisiae 35S pre-rRNA gene under the control of the galactose-inducible GAL7 promoter (Beltrame and Tollervey, 1992; Henry et al, 1994) (Figure 4A). To facilitate monitoring of 18S and 25S expression from the plasmid-borne rDNA allele, neutral sequence tags had been inserted into the 5′-terminal regions of the 18S and 25S rRNA genes. Moreover, we introduced nucleotide changes into the rm1 and rm2 motifs of the 18S rRNA gene that reduced the potential of the resulting 18Srm1 (pTH25rm1) and 18Srm2 (pTH25rm2) rRNAs for base pairing with the m1 and m2 motifs of snR30, respectively.

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