PIN domain of Nob1p is required for D-site cleavage in 20S pre-rRNA

Fatica, Alessandro; Tollervey, David; Dlakić, Mensur

doi:10.1261/rna.7123504

Cited by 116 publications

(133 citation statements)

References 38 publications

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“…24,34 The characterized eukaryotic PIN domain proteins are involved in the process of nonsense codon mediated decay (NMD) of mRNA 19,35 and processing of pre-18S rRNA. 36 In archaea a single group of conserved PIN domains are found in an operon with other ribosomal and translation factor genes, suggesting that this version, 34 like its eukaryotic counter-part, is also involved in processing of pre-rRNAs. Given that the operons allow clear prediction of the functional associations of PIN domains in prokaryotes, we investigated if contextual information might similarly throw light on the functions of the prokaryotic NYN domains.…”

Section: Resultsmentioning

confidence: 99%

The NYN Domains: Novel Predicted RNAses with a PIN Domain-Like Fold

Anantharaman¹,

Aravind²

2006

RNA Biology

124

155

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Using sensitive sequence profile searches and contextual information gleaned from domain architectures and predicted operons we identify a novel family of protein domains with predicted ribonuclease activity. These domains are found in the eukaryotic proteins typified by the Nedd4-binding protein 1 and the bacterial YacP-like proteins (Nedd4-BP1, YacP nucleases; NYN domains). We show that the NYN domain shares a common protein fold with two other previously characterized groups of nucleases, namely the PIN (PilT N-terminal) and FLAP/5' → 3' exonuclease superfamilies. We also show that all these proteins share a common set of 4 acidic conserved residues that are predicted to constitute their active site. Based on the conservation of the acidic residues and structural elements we suggest that PIN and NYN domains are likely to bind only a single metal ion, unlike the FLAP/5' → 3' exonuclease superfamily, which binds two metal ions. We also present evidence that the other conserved residues shared by all these three domains are likely to play critical roles in sensing the substrate and positioning the catalytic residues in the right conformation. Based on conserved gene neighborhoods we infer that the bacterial members are likely to be components of the processome/ degradsome that process tRNAs or ribosomal RNAs. Eukaryotic versions appear to have undergone extensive functional diversification as suggested by the several distinctive multi-domain architectures showing fusions with various other RNA-binding domains like CCCH, PPR and KH domains. Interestingly, the eukaryotic NYN domains also show multiple fusions to the UBA domain, an ubiquitin-binding adaptor domain. This observation, together with the mono-ubiquitination of Nedd4-BP1 by the ubiquitin ligase Nedd4 suggests that the NYN domain proteins of eukaryotes are regulated by mono-ubiquitination. Given the localization of Nedd4-BP1 to punctuate nuclear bodies, it is likely that they are parts of nuclear RNA-processing complexes that are dependent on mono-ubiquitination for their assembly.

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Section: Resultsmentioning

confidence: 99%

The NYN Domains: Novel Predicted RNAses with a PIN Domain-Like Fold

Anantharaman¹,

Aravind²

2006

RNA Biology

124

155

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“…Consistent with this function, Nob1 is not associated with the SSU processome͞90S preribosome but instead is a component of the 43S preribosome (20). Interestingly, of the two mutations in the predicted active site of Nob1, one abolished Nob1 protein function in vivo (9). Therefore, Nob1 was suggested to be the endonuclease that cleaves the 20S pre-rRNA at site D. However, the proposed nuclease activity of Nob1 has so far not been demonstrated in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…Only three have been previously characterized, including Nmd4, a protein involved in nonsense-mediated mRNA decay, Rrp44, an exosome component, and Nob1. Nob1 is a component of the 43S preribosome and has been suggested to be the endonuclease that cleaves the 20S pre-rRNA at site D in the cytoplasm (8,9). Although the nuclease function of eukaryotic PIN domain proteins has not yet been demonstrated in vitro, the function of a subset of them in RNA metabolism and the conservation of the putative active site residues make them attractive candidates.…”

mentioning

confidence: 99%

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Bleichert

Granneman

Osheim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

104

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Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A 1 and͞or A2.ribosome biogenesis ͉ small subunit processome ͉ endonuclease ͉ yeast ͉ RNA processing R ibosomes translate mRNA into proteins and, thereby, govern cell growth and survival. In Saccharomyces cerevisiae, ribosome biogenesis starts with the transcription of the rDNA into a polycistronic rRNA precursor by RNA polymerase I in the nucleolus. The primary transcript, the 35S pre-rRNA, encodes the small subunit (SSU) rRNA, 18S, the large subunit rRNAs, 25S and 5.8S, and also external and internal transcribed spacer regions (5ЈETS, ITS1, ITS2, and 3ЈETS; Fig. 1). This precursor is extensively endoand exonucleolytically cleaved to mature the rRNAs. Maturation begins with endonucleolytic cleavages at sites A 0 , A 1 , and A 2 , the latter separating the 20S pre-rRNA, the direct precursor to the mature 18S rRNA, and the 27SA 2 pre-rRNA, the precursor to the large subunit rRNAs. The 20S pre-rRNA is exported into the cytoplasm as part of the 43S preribosome, where the 18S rRNA is matured by cleavage at site D.The A 0 , A 1 , and A 2 pre-rRNA cleavages require a large ribonucleoprotein complex, the SSU processome͞90S preribosome, which contains at least 40 different proteins and numerous small nucleolar RNAs (snoRNAs) (1-4). This complex likely corresponds to the terminal knobs seen in Miller chromatin spreads (1, 5). Depletion of essential SSU processome components prevents cleavages at sites A 0 -A 2 but not A 3 . Although this results in accumulation of the 23S pre-rRNA and inhibition of 18S synthesis, maturation of the 25S and 5.8S rRNAs can occur normally (Fig. 1).Despite the identification of numerous proteins involved in early pre-18S rRNA processing by large-scale purifications, the endonuclease(s) and possible candidates that carry out these cleavages have remained elusive. Recently, an interesting group of proteins possessing a PIN domain (PilT N terminus, PINc domain in SMART database) has been impl...

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“…7A). As a control, we also affinity-purified a mutant Nob1-D15N protein, in which the catalytic nuclease activity of the PIN domain was predicted to be destroyed (10,11). Since the Nob1-D15N mutant on its own does not support growth (Fig.…”

Section: A Genetic Network Between Thementioning

confidence: 99%

RNA Helicase Prp43 and Its Co-factor Pfa1 Promote 20 to 18 S rRNA Processing Catalyzed by the Endonuclease Nob1

Pertschy

Schneider²,

Gnädig³

et al. 2009

Journal of Biological Chemistry

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177

287

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Many RNA nucleases and helicases participate in ribosome biogenesis, but how they cooperate with each other is largely unknown. Here we report that in vivo cleavage of the yeast pre-rRNA at site D, the 3-end of the 18 S rRNA, requires functional interactions between PIN (PilT N terminus) domain protein Nob1 and the DEAH box RNA helicase Prp43. Nob1 showed specific cleavage on a D-site substrate analogue in vitro, which was abolished by mutations in the Nob1 PIN domain or the RNA substrate. Genetic analyses linked Nob1 to the late pre-40 S-associated factor Ltv1, the RNA helicase Prp43, and its cofactor Pfa1. In strains lacking Ltv1, mutation of Prp43 or Pfa1 led to a striking accumulation of 20 S pre-rRNA in the cytoplasm due to inhibition of site D cleavage. This phenotype was suppressed by increased dosage of wildtype Nob1 but not by Nob1 variants mutated in the catalytic site. In ltv1/pfa1 mutants the 20 S pre-rRNA was susceptible to 3 to 5 degradation by the cytoplasmic exosome. This degraded into the 3 region of the 18 S rRNA, strongly indicating that the preribosomes are structurally defective.Eukaryotic ribosome formation is a complex process that requires coordination of rRNA synthesis and processing with the subsequent incorporation of the ribosomal (Rpl and Rps) proteins (reviewed in Refs. 1-5). In the yeast Saccharomyces cerevisiae, ribosome synthesis starts with the transcription of a 35 S pre-rRNA, which is the common precursor to the mature 18, 5.8, and 25 S rRNAs. This pre-rRNA is co-transcriptionally bound by ribosomal proteins as well as many non-ribosomal trans-acting factors to form a 90 S preribosomal particle. Next, this 90 S intermediate undergoes a series of early pre-rRNA processing and modification steps in the nucleolus before an endonucleolytic cleavage at site A 2 in the pre-rRNA separates the pathways for 60 and 40 S subunit assembly.Pre-60 S particles undergo further maturation steps in the nucleus, whereas pre-40 S particles contain few non-ribosomal proteins and are rapidly exported to the cytoplasm. The factors involved in 40 S export are largely unknown, but the non-essential pre-40 S factor Ltv1 was suggested to play a role in this process (6). Following nuclear export, the pre-40 S subunit undergoes cytoplasmic maturation, involving two major events. (i) 20 S pre-rRNA cleavage at site D generates the 3Ј-end of the mature 18 S rRNA, and (ii) structural reorganization forms the characteristic beak structure of the mature 40 S subunit, resulting in exposure of RNA helix 33 within the 18 S rRNA (7).The sites and the order of endonuclease and exonuclease processing events that convert the 35 S pre-rRNA into the mature rRNA species are well characterized in yeast (Fig. 3A) (reviewed in Refs. 4 and 8). However, although most or all of the exonucleases participating in rRNA processing are known, several predicted endonucleases remain to be identified. Two proteins, Fap7 and Nob1, were proposed to cleave site D (9 -11), but for neither was endonuclease activity shown. Fap7 is a putativ...

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PIN domain of Nob1p is required for D-site cleavage in 20S pre-rRNA

Cited by 116 publications

References 38 publications

The NYN Domains: Novel Predicted RNAses with a PIN Domain-Like Fold

The NYN Domains: Novel Predicted RNAses with a PIN Domain-Like Fold

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

RNA Helicase Prp43 and Its Co-factor Pfa1 Promote 20 to 18 S rRNA Processing Catalyzed by the Endonuclease Nob1

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