rRNA Maturation as a “Quality” Control Step in Ribosomal Subunit Assembly in Dictyostelium discoideum

Mangiarotti, G; Chiaberge, Sara; Bulfone, S.

doi:10.1074/jbc.272.44.27818

Cited by 14 publications

(13 citation statements)

References 39 publications

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“…Since this structure lacks initiator tRNA, it was proposed that it is not engaged in translation and that its disassembly would be required before 40S mRNA binding, reassembly, and productive translation initiation (Strunk et al 2012). Earlier studies in Dictyostelium discoideum had reached a similar conclusion but had more radically proposed that 18S maturation occurs as in Escherichia coli primarily after 80S formation, possibly at the first translation initiation stage (Mangiarotti et al 1997;Shajani et al 2011). Therefore, although for many years it has been assumed that incomplete 18S processing prevents nuclear subunits from associating, there is now abundant evidence that the 40S subunit containing 20S pre-rRNA can interact with 60S.…”

Section: Introductionmentioning

confidence: 67%

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

Al-Jubran

Wen

Abdullahi

et al. 2013

RNA

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In eukaryotes the 40S and 60S ribosomal subunits are assembled in the nucleolus, but there appear to be mechanisms preventing mRNA binding, 80S formation, and initiation of translation in the nucleus. To visualize association between ribosomal subunits, we tagged pairs of Drosophila ribosomal proteins (RPs) located in different subunits with mutually complementing halves of fluorescent proteins. Pairs of tagged RPs expected to interact, or be adjacent in the 80S structure, showed strong fluorescence, while pairs that were not in close proximity did not. Moreover, the complementation signal is found in ribosomal fractions and it was enhanced by translation elongation inhibitors and reduced by initiation inhibitors. Our technique achieved 80S visualization both in cultured cells and in fly tissues in vivo. Notably, while the main 80S signal was in the cytoplasm, clear signals were also seen in the nucleolus and at other nuclear sites. Furthermore, we detected rapid puromycin incorporation in the nucleolus and at transcription sites, providing an independent indication of functional 80S in the nucleolus and 80S association with nascent transcripts.

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Section: Introductionmentioning

confidence: 67%

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

Al-Jubran

Wen

Abdullahi

et al. 2013

RNA

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“…This possibility is strengthened by the observation that in normally growing cells, Nob1, Fap7, and a small amount of the 20S pre-rRNA cosediment with 80S ribosomes but not with polyribosomes (6 and this work). Furthermore, Dictyostelium discoideum preribosomes are often detected in 80S monosomes and even polyribosomes in vivo and in vitro (31,32). In this organism, the assembly of immature ribosomal subunits into polyribosomes is an obligatory step, and it has been shown that if newly formed ribosomes cannot function in translation, late rRNA maturation steps do not take place (31).…”

Section: Discussionmentioning

confidence: 99%

The Putative NTPase Fap7 Mediates Cytoplasmic 20S Pre-rRNA Processing through a Direct Interaction with Rps14

Granneman¹,

Nandineni²,

Baserga

2005

Molecular and Cellular Biology

124

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One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3 end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.In eukaryotes, rRNA transcription and ribosome biogenesis occur in a subnuclear compartment called the nucleolus. In this subcompartment, RNA polymerase I transcribes an rRNA precursor (pre-rRNA) that harbors the 18S, 25S/28S, and 5.8S rRNAs and several noncoding internal and external transcribed spacers (ITS and ETS, respectively) ( Fig. 1). The prerRNA is chemically modified and cleaved by endo-and exonucleases to produce the mature rRNAs. This process has been most extensively characterized in the yeast Saccharomyces cerevisiae (for detailed reviews, see references 36 and 46). In this organism, the primary 35S pre-rRNA is cleaved at sites A 0 , A 1 , and A 2 to yield the 20S and 27SA 2 pre-rRNA intermediates. These cleavage steps are mediated by components of the ϳ80S small-subunit (SSU) processome/90S preribosomes (4, 18). The 20S pre-rRNA, packaged into 43S preribosomes, is exported to the cytoplasm, where it is dimethylated by Dim1 and processed at site D to form the mature 18S rRNA and thereby the 40S ribosomal subunit (SSU). The 27SA 2 pre-rRNAs, part of 66S preribosomes, can be processed via two pathways leading to the synthesis of the 5.8S and 25S large-subunit (LSU) rRNAs (Fig. 1). Finally, the 5S rRNA is independently transcribed as a precursor by RNA polymerase III (Fig. 1). Many of the cleavage steps in pre-rRNA processing are believed to be endonucleolytic; thus far, however, the enzymes responsible for most of these cleavages have not been identified. Two well-studied examples are the RNase MRP snoRNP, which cleaves at site A 3 , and Rnt1, an endonuclease responsible for cleavage of the 3Ј ETS (24, 30). One possible candidate for the cleavage at site D in the 20S pre-rRNA is Nob1, a protein that contains a putative PIN domain, which shares structural homology with several exonucleases and flap endonucleases (2,6,8). Consistent with a role as a nuclease, conserved residues within...

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“…Based on these observations, it is conceivable that 20 to 18 S rRNA processing might occur after 40 S-60 S subunit joining or even after the first round of translation. In Dictyostelium, only 40 S subunits that are capable of translation undergo the final rRNA processing step, providing a precedent (51).…”

Section: Discussionmentioning

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

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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rRNA Maturation as a “Quality” Control Step in Ribosomal Subunit Assembly in Dictyostelium discoideum

Cited by 14 publications

References 39 publications

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

The Putative NTPase Fap7 Mediates Cytoplasmic 20S Pre-rRNA Processing through a Direct Interaction with Rps14

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

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