Based on the results of a high-throughput proteomic analysis, we speculated that Nob1p might be present in pre-40S ribosomes, and we show here that this is indeed the case. On sucrose gradients, the major peak of Nob1-TAP cosedimented with pre-40S particles, with a minor peak at the position of 90S preribosomes. Northern and primer extension analyses of immunoprecipitated RNAs confirmed the association of Nob1p with the 20S pre-rRNA, as well as a weaker association with the 35S pre-rRNA and U3 small nucleolar RNA components of the 90S preribosomes. Moreover, the precipitated 20S pre-rRNA was shown to be dimethylated within the 3′ region of the 18S rRNA. This modification is reported to occur in the cytoplasm (4
), indicating that Nob1p accompanies the 20S pre-rRNA from the nucleus to the cytoplasm. Consistent with this proposal, Nob1-TAP was localized in both the nucleus and cytoplasm.
Depletion of Nob1p led to the loss of 18S rRNA synthesis, accompanied by the accumulation of remarkably high levels of the 20S pre-rRNA. In the absence of correct ribosome synthesis, the preribosomal particles are generally degraded rapidly. Only in a very few mutant strains do the pre-rRNAs accumulate to a level that represents a significant fraction of the abundance of the mature rRNAs. The ready detection of the 20S pre-rRNA by ethidium staining in the Nob1p-depleted strain is therefore an unusual feature, which might be more consistent with the specific inhibition of pre-rRNA cleavage than with a general defect in pre-40S structure. Together with the presence of the PIN domain in Nob1p, this would be consistent with a direct role in pre-rRNA processing.
A partial processing defect was seen in GAL::nob1 strains grown on permissive, galactose medium. We speculate that excess Nob1p competes for binding to other components of the 20S processing complex, delaying its productive assembly onto the pre-rRNA.
In the Nob1p-depleted strain, 20S pre-rRNA degradation products were also readily observed. The accumulation of such readily detectable degradation intermediates is also unusual in strains that are not compromised in RNA turnover systems. The major species extended from within the 3′ region of the 18S rRNA to the 3′ end of the 20S pre-rRNA (site A2 in ITS1). It is unclear whether the 5′ ends of these species are generated by inaccurate 18S cleavage in the absence of Nob1p or by pausing of 5′ → 3′ exonucleases upstream of site D. In either case, we speculate that the accumulation of these fragments may be a consequence of the assembly of a nonproductive processing complex at the 3′ end of the 18S rRNA and on the 5′ region of ITS1. The size of the largest of these species indicates that the 5′ end is positioned close to the site of m26Am26A dimethylation in the 3′-terminal loop of the 18S rRNA. This modification imposes a complete block on the progression of reverse transcriptase. The effects of RNA modification on the activity of Xrn1p have not been assessed, but pausing at this site during 5′ degradation of the 20S pre-rRNA is an alternative possibility.
Nob1p was identified as a protein that interacted with Nin1p, a regulatory component of the 26S proteosome (34
), and it is reported to be required for proteosome biogenesis (35
). Consistent with this, Nob1p was reported to sediment at 26S on a sucrose gradient, based on Western analyses with anti-Nob1p (34
). We did not, however, detect TAP-tagged Nob1p at this gradient position, and inspection of the published data suggests that Nob1p might have been misidentified in the previous analysis. An additional protein band was decorated by anti-Nob1p that showed gel mobility appropriate for Nob1p and sedimented at approximately 40S on the gradient (34
). The data therefore do not support a stable interaction between Nob1p and the 26S proteosome, consistent with the report that Nob1p is degraded following proteosome synthesis (35
A possible functional interaction between Nob1p and the proteosome can be envisaged in ribosome synthesis. The pre-rRNAs present in defective preribosomal particles are normally very rapidly degraded, but the fate of the associated ribosomal proteins is not clear. The Nob1p-Nin1p interaction might promote the recruitment of the proteosome to ribosomal subunits that are stalled in maturation, in order to bring about r-protein degradation.