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Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis.

Bohnsack MT, Martin R, Granneman S, Ruprecht M, Schleiff E, Tollervey D - Mol. Cell (2009)

Bottom Line: In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association.Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions.We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK. bohnsack@bio.uni-frankfurt.de

ABSTRACT
Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3' cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.

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Prp43 Crosslinking to snoRNAs and snRNAsProtein-RNA complexes from Prp43-HTP-expressing cells and control cells were crosslinked and analyzed as described for Figure 1. The percentage of sequences found for individual snoRNAs (A) or snRNAs (B) and the crosslinking sites of Prp43 on the most highly enriched snoRNAs snR51 (C), snR72 (D), snR60 (E), and the splicing snRNA U6 (F) are presented as total number of hits. The total number of hits (red line) and the positions of deletions (dashed green line) and nucleotide substitutions (dashed blue line) in the obtained sequences are shown. The regions of the guide sequences and functional elements of the snoRNAs are indicated below.
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fig3: Prp43 Crosslinking to snoRNAs and snRNAsProtein-RNA complexes from Prp43-HTP-expressing cells and control cells were crosslinked and analyzed as described for Figure 1. The percentage of sequences found for individual snoRNAs (A) or snRNAs (B) and the crosslinking sites of Prp43 on the most highly enriched snoRNAs snR51 (C), snR72 (D), snR60 (E), and the splicing snRNA U6 (F) are presented as total number of hits. The total number of hits (red line) and the positions of deletions (dashed green line) and nucleotide substitutions (dashed blue line) in the obtained sequences are shown. The regions of the guide sequences and functional elements of the snoRNAs are indicated below.

Mentions: Small nuclear RNA sequences obtained after in vivo crosslinking of Prp43-HTP expressing cells or controls are shown as heat maps, indicating the percentage of mapped reads for each snoRNA (Figure 3A) or snRNA (Figure 3B). In vitro crosslinking of Prp43-containing complexes did not significantly enrich for small noncoding RNAs (Table S2).


Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis.

Bohnsack MT, Martin R, Granneman S, Ruprecht M, Schleiff E, Tollervey D - Mol. Cell (2009)

Prp43 Crosslinking to snoRNAs and snRNAsProtein-RNA complexes from Prp43-HTP-expressing cells and control cells were crosslinked and analyzed as described for Figure 1. The percentage of sequences found for individual snoRNAs (A) or snRNAs (B) and the crosslinking sites of Prp43 on the most highly enriched snoRNAs snR51 (C), snR72 (D), snR60 (E), and the splicing snRNA U6 (F) are presented as total number of hits. The total number of hits (red line) and the positions of deletions (dashed green line) and nucleotide substitutions (dashed blue line) in the obtained sequences are shown. The regions of the guide sequences and functional elements of the snoRNAs are indicated below.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806949&req=5

fig3: Prp43 Crosslinking to snoRNAs and snRNAsProtein-RNA complexes from Prp43-HTP-expressing cells and control cells were crosslinked and analyzed as described for Figure 1. The percentage of sequences found for individual snoRNAs (A) or snRNAs (B) and the crosslinking sites of Prp43 on the most highly enriched snoRNAs snR51 (C), snR72 (D), snR60 (E), and the splicing snRNA U6 (F) are presented as total number of hits. The total number of hits (red line) and the positions of deletions (dashed green line) and nucleotide substitutions (dashed blue line) in the obtained sequences are shown. The regions of the guide sequences and functional elements of the snoRNAs are indicated below.
Mentions: Small nuclear RNA sequences obtained after in vivo crosslinking of Prp43-HTP expressing cells or controls are shown as heat maps, indicating the percentage of mapped reads for each snoRNA (Figure 3A) or snRNA (Figure 3B). In vitro crosslinking of Prp43-containing complexes did not significantly enrich for small noncoding RNAs (Table S2).

Bottom Line: In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association.Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions.We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK. bohnsack@bio.uni-frankfurt.de

ABSTRACT
Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3' cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.

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