Nob1p Is Required for Cleavage of the 3′ End of 18S rRNA

Abstract

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.Eukaryotic ribosome synthesis takes place largely within the nucleolus, but the late steps in both pre-40S and pre-60S maturation occur after export of the subunits to the cytoplasm. In Saccharomyces cerevisiae, this cytoplasmic maturation includes an endonuclease cleavage at site D, the 3Ј end of the 18S rRNA that generates the mature rRNA from the 20S prerRNA ( Fig. 1) (27,37). Around 150 proteins and 80 small nucleolar RNAs are known to be required for the posttranscriptional steps in yeast ribosome synthesis. Despite the identification of this large number of factors, there are a great many gaps in our knowledge. Among these are the identities of the endonucleases that perform several of the pre-rRNA cleavages including that at the 3Ј end of the mature 18S rRNA (Fig. 1). The pathway of ribosome subunit synthesis in yeast was initially studied by sucrose gradient centrifugation (16,36,38), leading to the identification of a 90S particle that contains the 35S pre-rRNA, a 66S particle that contains the 27S prerRNAs, and a 43S particle containing the 20S pre-rRNA. Recent analyses have shown that the precursors to the 60S particle are substantially more complex than was initially proposed, with multiple pre-60S particles containing different complements of the precursors to the 25S and 5.8S rRNAs and different protein compositions (6, 13, 28, 31; reviewed in references 7 and 41). Equivalent analyses of the pre-40S ribosomes have not yet been reported, but a high-throughput proteomic analysis of yeast protein interactions identified three complexes that potentially represented such particles (8; reviewed in reference 7), one of which was associated with tandem affinity purification (TAP)-tagged Nob1p (Yor056c).Each of these complexes contained Tsr1p, which is required for 20S-to-18S processing (9), and one also contained the dimethylase Dim1p, which modifies the 20S pre-rRNA, probably in the cytoplasm, and is additionally required for nucleolar pre-rRNA processing (4,19,22). Rrp10p (Rio1p) is also required for 20S-to-18S processing (39) but was not identified in these putative pre-40S particles.Nob1p (for "Nin one binding protein") was initially reported to interact in a two-hybrid screen with Nin1p/Rpn12p, a subunit of the 19S regulatory particle of the yeast 26S proteosom...

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

“…The early 43S preribosomal particle is rapidly exported to the cytoplasm, where the conversion of 20S pre-rRNA into mature 18S rRNA and the last assembly reactions take place (Udem and Warner 1973). The study of the different purified pre-40S complexes indicates that only few transacting factors are needed to finish the maturation of 40S r-subunits from early 43S preribosomal particles (Schäfer et al 2002;Fatica et al 2003a;Vanrobays et al 2003). However, the study of the different purified pre-60S complexes is consistent with the presence of distinct pre-60S intermediates that move from the nucleolus to the nucleoplasm and from there to the cytoplasm.…”

Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA–protein interactions during small ribosomal subunit biogenesis

“…The pre-SSU binds Nob1 already in the nucleus, but D-site cleavage occurs only after its export into the cytoplasm (15). Depletion of Nob1 leads to the accumulation of 20S pre-rRNA containing SSU precursors (13,14). Interestingly, depletion of Fap7, mutations in its Walker A and B motifs (10), or mutations in the arginine-rich C-terminal tail of Rps14 also lead to accumulation of 20S prerRNA (16).…”

“…When Fap7 is depleted in yeast or mutated in its Walker B motif, the nascent SSU lacks a third ribosomal protein, Rps14 (12). It also contains a 20S pre-rRNA, which requires cleavage by Nob1 at site D to yield mature 18S rRNA (13,14). The pre-SSU binds Nob1 already in the nucleus, but D-site cleavage occurs only after its export into the cytoplasm (15).…”

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

“…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.…”