Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

Bousquet‐Antonelli, Cécile; Vanrobays, Emmanuel; Gélugne, Jean‐Paul; Caizergues‐Ferrer, Michèle; Henry, Y.

doi:10.1017/s1355838200992288

Cited by 44 publications

(48 citation statements)

References 79 publications

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“…Y0341-npa1::GFP cells were treated as previously described (10). Detection of Npa1p-ZZ by immunoelectron microscopy was performed as described by Henras et al (38).…”

Section: Methodsmentioning

confidence: 99%

“…A total of 500 l of extract corresponding to 5 mg of proteins was loaded on a 10 to 30% glycerol gradient. Preparation of the gradient, loading of the extract, centrifugation, and collection of fractions were performed as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…RNA fractionations by agarose or polyacrylamide gel electrophoresis were performed as described by Henras et al (38). Primer extensions were performed as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

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Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification

Dez

Froment

Noaillac-Depeyre

et al. 2004

Molecular and Cellular Biology

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105

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We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.Synthesis of the 40S and 60S ribosomal subunits in eukaryotes is a particularly intricate process that involves the synthesis of four ribosomal RNAs, their assembly with close to 80 ribosomal proteins, and transport of preribosomal particles from the nucleus to the cytoplasm where translation occurs (30,56,76,98). Eukaryotic ribosome synthesis requires the action of RNA polymerases I, II, and III that, respectively, transcribe a long common precursor to the 18S, 5.8S, and 25S rRNAs, the pre-mRNAs of ribosomal proteins, and the precursor to 5S rRNA. The pre-rRNA transcribed by RNA polymerase I contains, in addition to the sequences retained in the mature cytoplasmic ribosomes, long spacer regions that will be removed by a complex series of endo-and exonucleolytic cleavage steps (for a schematic representation of the pre-rRNA processing steps, see Fig. 4C). Moreover, specific nucleotides of rRNAs will undergo posttranscriptional chemical modification. By far the two most common modifications are the conversion of uridines into pseudouridines and the methylation of the oxygen at the 2Ј position of ribose moieties. These modifications are carried out by box H/ACA and box C/D small nucleolar RNPs (snoRNPs), respectively (4,26,28,49,50,86). Although the precise functions of modified nucleotides in rRNAs are not known, they are believed to significantly contribute to ribosome function since they are present in the most highly conserved and functionally important regions of rRNAs (15). Indeed, M. Fournier and collaborators have recently shown that lack of a single pseudouridine within the peptidyl transferase center, due to alteration of the box H/ACA snoRNP that produces this pseudouridine, is correlated with a substantial reduction in translational activity (48). Likewise, Bonnerot and colleagues have shown that the absence of 2Ј-O-ribose methylation of U2918 within the peptidyl transferase center increases sensitivity to paro...

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“…Y0341-npa1::GFP cells were treated as previously described (10). Detection of Npa1p-ZZ by immunoelectron microscopy was performed as described by Henras et al (38).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification

Dez

Froment

Noaillac-Depeyre

et al. 2004

Molecular and Cellular Biology

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105

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“…This may be due to the transient nature of these interactions or concurrent disassembly of the SSU processome when Bud23 enters the particle. The behavior of Bud23 is reminiscent of another putative methyltransferase, Rrp8, that shows synthetic lethality with gar1 mutants and strongly affects cleavage at A2 but does not immunoprecipitate SSU processome components or H/ACA or C/D box snoRNPs (Bousquet-Antonelli et al 2000). However, how Rrp8 mediates its role in rRNA processing or its putative substrate is not understood.…”

Section: Bud23 Work At the Transition Of Early To Middle Stage Of 40mentioning

confidence: 99%

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

Sardana

White

Johnson

2013

RNA

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Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

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“…Yeast Gar1p, Nhp2p, Nop10p, Nop1p, ZZ-tagged proteins, and hNop10 were detected as described in Bousquet-Antonelli et al (2000), Henras et al (2001), and Dez et al (2002). Yeast Cbf5p was detected using purified antibodies, diluted 500-fold, raised in rabbits (by Eurogentec) against the recombinant protein lacking the C-terminal KKE repeats.…”

Section: Western Analysismentioning

confidence: 99%

hNaf1 is required for accumulation of human box H/ACA snoRNPs, scaRNPs, and telomerase

Hoareau-Aveilla¹,

Bonoli²,

Caizergues‐Ferrer³

et al. 2006

RNA

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The human telomerase ribonucleoprotein particle (RNP) shares with box H/ACA small Cajal body (sca)RNPs and small nucleolar (sno)RNPs the proteins dyskerin, hGar1, hNhp2, and hNop10. How dyskerin, hGar1, hNhp2, and hNop10 assemble with box H/ACA scaRNAs, snoRNAs, and the RNA component of telomerase (hTR) in vivo remains unknown. In yeast, Naf1p interacts with H/ACA snoRNP proteins and may promote assembly of Cbf5p (the yeast ortholog of dyskerin) with nascent presnoRNAs. Here we show that the human HsQ96HR8 protein, thereafter termed hNaf1, can functionally replace endogenous Naf1p in yeast. HeLa hNaf1 associates with dyskerin and hNop10 as well as box H/ACA scaRNAs, snoRNAs, and hTR. Reduction of hNaf1 steady-state levels by RNAi significantly lowers accumulation of these components of box H/ACA scaRNP, snoRNP, and telomerase. hNaf1 is found predominantly in numerous discrete foci in the nucleoplasm and fails to accumulate within Cajal bodies or nucleoli. Altogether, these results suggest that hNaf1 intervenes in early assembly steps of human box H/ACA RNPs, including telomerase.

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Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

Cited by 44 publications

References 79 publications

Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification

Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

hNaf1 is required for accumulation of human box H/ACA snoRNPs, scaRNPs, and telomerase

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