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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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Identification of Putative Binding Sites of Prp43 on Ribosomal RNAControl cells (C) or Prp43-HTP-expressing cells (A) were UV crosslinked in vivo or crosslinking was performed in vitro after cell lysis and enrichment of Prp43-HTP-containing complexes on IgG Sepharose (B). Crosslinked RNAs were trimmed and ligated to linkers followed by RT-PCR and Solexa sequencing. The obtained sequences were aligned with the rDNA encoding 35S pre-rRNA (nucleotides 1–6880), and the number of hits for each individual nucleotide is plotted. The position of the mature 18S, 5.8S, and 25S rRNAs is indicated by bars below, and processing site D is shown. Numbers at peaks indicate the corresponding helices of 18S or 25S, while 25S peaks labeled with “C” represent background peaks also found in the control. In vivo crosslinking of Prp43-HTP-expressing cells resulted in two major peaks, whereas in vitro crosslinking of purified complexes yielded three sequence clusters. Thus, CRAC experiments have identified several potential binding sites for Prp43 on pre-rRNA.
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fig1: Identification of Putative Binding Sites of Prp43 on Ribosomal RNAControl cells (C) or Prp43-HTP-expressing cells (A) were UV crosslinked in vivo or crosslinking was performed in vitro after cell lysis and enrichment of Prp43-HTP-containing complexes on IgG Sepharose (B). Crosslinked RNAs were trimmed and ligated to linkers followed by RT-PCR and Solexa sequencing. The obtained sequences were aligned with the rDNA encoding 35S pre-rRNA (nucleotides 1–6880), and the number of hits for each individual nucleotide is plotted. The position of the mature 18S, 5.8S, and 25S rRNAs is indicated by bars below, and processing site D is shown. Numbers at peaks indicate the corresponding helices of 18S or 25S, while 25S peaks labeled with “C” represent background peaks also found in the control. In vivo crosslinking of Prp43-HTP-expressing cells resulted in two major peaks, whereas in vitro crosslinking of purified complexes yielded three sequence clusters. Thus, CRAC experiments have identified several potential binding sites for Prp43 on pre-rRNA.

Mentions: Ribosomal sequences obtained by CRAC were aligned against the rDNA, and the number of hits was determined for each individual nucleotide. Peaks represent clusters of hits covering the same sequence and are therefore potential binding sites for Prp43 (Figure 1). In total, five major peaks were identified by in vivo and in vitro crosslinking (see the supplemental sequence alignments in the Supplemental Data). In addition, multiple smaller clusters were found, some of which were also present in the control. A very similar distribution of hits was obtained by cloning and Sanger sequencing of the RT-PCR products (data not shown). In vivo crosslinking of Prp43-HTP enriched two major regions relative to the control (Figure 1A). A bipartite peak was observed over rDNA nucleotides 2440 and 2470, corresponding to sites within helix 44 near the 3′ end of 18S (Figure 2A). These sites are located close to pre-rRNA cleavage site D, where processing of the 20S pre-rRNA to the mature 18S rRNA occurs. This observation is consistent with the model that Prp43 acts to promote pre-rRNA cleavage at site D by the endonuclease Nob1 (Pertschy et al., 2009). One site of RNA 2′-O-methylation is located within this region, which is directed by snR70 (Figure 2A). The other major in vivo peak was located over rDNA nucleotides 4427-4440, which was centered at 25S+1145, between helices 39 and 40. This 25S peak is located close to the 2′-O-methylation site (Am1133) directed by the box C/D snoRNP snR61 and to the pseudouridylation site (Ψ1124) directed by box H/ACA snoRNP snR5 (Figure 2B).


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)

Identification of Putative Binding Sites of Prp43 on Ribosomal RNAControl cells (C) or Prp43-HTP-expressing cells (A) were UV crosslinked in vivo or crosslinking was performed in vitro after cell lysis and enrichment of Prp43-HTP-containing complexes on IgG Sepharose (B). Crosslinked RNAs were trimmed and ligated to linkers followed by RT-PCR and Solexa sequencing. The obtained sequences were aligned with the rDNA encoding 35S pre-rRNA (nucleotides 1–6880), and the number of hits for each individual nucleotide is plotted. The position of the mature 18S, 5.8S, and 25S rRNAs is indicated by bars below, and processing site D is shown. Numbers at peaks indicate the corresponding helices of 18S or 25S, while 25S peaks labeled with “C” represent background peaks also found in the control. In vivo crosslinking of Prp43-HTP-expressing cells resulted in two major peaks, whereas in vitro crosslinking of purified complexes yielded three sequence clusters. Thus, CRAC experiments have identified several potential binding sites for Prp43 on pre-rRNA.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Identification of Putative Binding Sites of Prp43 on Ribosomal RNAControl cells (C) or Prp43-HTP-expressing cells (A) were UV crosslinked in vivo or crosslinking was performed in vitro after cell lysis and enrichment of Prp43-HTP-containing complexes on IgG Sepharose (B). Crosslinked RNAs were trimmed and ligated to linkers followed by RT-PCR and Solexa sequencing. The obtained sequences were aligned with the rDNA encoding 35S pre-rRNA (nucleotides 1–6880), and the number of hits for each individual nucleotide is plotted. The position of the mature 18S, 5.8S, and 25S rRNAs is indicated by bars below, and processing site D is shown. Numbers at peaks indicate the corresponding helices of 18S or 25S, while 25S peaks labeled with “C” represent background peaks also found in the control. In vivo crosslinking of Prp43-HTP-expressing cells resulted in two major peaks, whereas in vitro crosslinking of purified complexes yielded three sequence clusters. Thus, CRAC experiments have identified several potential binding sites for Prp43 on pre-rRNA.
Mentions: Ribosomal sequences obtained by CRAC were aligned against the rDNA, and the number of hits was determined for each individual nucleotide. Peaks represent clusters of hits covering the same sequence and are therefore potential binding sites for Prp43 (Figure 1). In total, five major peaks were identified by in vivo and in vitro crosslinking (see the supplemental sequence alignments in the Supplemental Data). In addition, multiple smaller clusters were found, some of which were also present in the control. A very similar distribution of hits was obtained by cloning and Sanger sequencing of the RT-PCR products (data not shown). In vivo crosslinking of Prp43-HTP enriched two major regions relative to the control (Figure 1A). A bipartite peak was observed over rDNA nucleotides 2440 and 2470, corresponding to sites within helix 44 near the 3′ end of 18S (Figure 2A). These sites are located close to pre-rRNA cleavage site D, where processing of the 20S pre-rRNA to the mature 18S rRNA occurs. This observation is consistent with the model that Prp43 acts to promote pre-rRNA cleavage at site D by the endonuclease Nob1 (Pertschy et al., 2009). One site of RNA 2′-O-methylation is located within this region, which is directed by snR70 (Figure 2A). The other major in vivo peak was located over rDNA nucleotides 4427-4440, which was centered at 25S+1145, between helices 39 and 40. This 25S peak is located close to the 2′-O-methylation site (Am1133) directed by the box C/D snoRNP snR61 and to the pseudouridylation site (Ψ1124) directed by box H/ACA snoRNP snR5 (Figure 2B).

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.

Show MeSH
Related in: MedlinePlus