Nop56p is the third common component of the large family of box C+D snoRNPs to be identified. ProtA-tagged Nop56p coprecipitated all tested box C+D snoRNAs with no significant coprecipitation of the box H+ACA snoRNAs or other small RNAs (MRP RNA and snRNAs). The highly charged carboxy-terminal KKD/E domain of Nop56p was not required for interactions with the snoRNAs. A similar carboxy-terminal truncation in Nop58p also had no effect on interaction with the snoRNAs (26
Previous studies indicated that Nop58p coprecipitated with all tested box C+D snoRNAs and was required for their accumulation (26
), showing it to be an integral component of all box C+D snoRNPs. Similarly, Nop1p coprecipitated with all tested box C+D snoRNAs (4
) and was required for the accumulation of all species expressed from pre-mRNA introns and polycistronic transcripts and must also be a component of the corresponding snoRNPs. Since Nop1p coprecipitated with Nop56p in stoichiometric amounts (15
) and these interactions did not take place in the absence of the snoRNAs, we conclude that Nop1p, Nop56p, and Nop58p are common components of most, and probably all, box C+D snoRNPs.
Based on our initial analysis of the in vivo assembly of the snoRNPs, we propose that Nop1p and Nop58p bind independently to the box C+D snoRNAs, with Nop56p then associating with Nop1p on the snoRNP. The association of Nop56p with the snoRNAs was only detected in the presence of Nop1p, whereas Nop58p could interact with the snoRNAs in the absence of Nop1p. Moreover, Nop1p and Nop58p are required for the stability of box C+D snoRNAs, whereas Nop56p is not, indicating that neither Nop1p nor Nop58p is dependent on Nop56p for association with the snoRNAs. This is consistent with the recent report that recombinant Xenopus
fibrillarin binds directly to the U16 snoRNA (11
) and indicates that Nop56p only interacts with snoRNAs that have prebound Nop1p. All tested box C+D snoRNAs were lost in strains with Nop58p depleted (26
), complicating the analysis of its role in snoRNP assembly. A 3′-extended form of U24 was, however, accumulated on depletion of Nop58p and was efficiently precipitated with ProtA-Nop56p, demonstrating that the association of Nop56p with this species at least was not dependent on Nop58p.
Most box C+D snoRNAs are synthesized by posttranscriptional processing, either from introns excised from pre-mRNAs or from polycistronic pre-snoRNAs. In both cases, 5′ processing of the mature snoRNAs involves the 5′→3′ exonucleases Rat1p and Xrn1p, while 3′ processing involves the exosome complex of 3′→5′ exonucleases (1
). All tested box C+D snoRNAs that are synthesized from introns or polycistronic transcripts were codepleted with Nop1p, with the exception of U14. Nop1p was not, however, required for normal accumulation of the U3 and snR4 snoRNAs that are encoded in monocistronic transcripts. This is distinct from the effects of Nop58p depletion, which strongly reduced the levels of all box C+D snoRNAs, including snR4, U3, and U14 (26
). In contrast, the levels of all tested snoRNAs were unaffected either by depletion of Nop56p or by the conditional-lethal nop56-2
Alterations in the position of the mature 3′ ends of several snoRNAs were seen in nop1
mutants. The mutant alleles of NOP1
uncoupled various defects in ribosome synthesis (43
) and also differ in their effects on snoRNA synthesis and accumulation. The strongest effects on 3′-end formation were shown by the nop1-5
strain at the permissive temperature (23°C), at which snoRNA accumulation was unaffected. Discrete 3′-extended forms of many snoRNAs are observed in strains lacking the Rrp6p component of the exosome complex, which is responsible for the final trimming of the pre-snoRNAs (2
). The extended snoRNAs in the nop1
mutants were similar in size, suggesting that Nop1p interacts with the exosome during snoRNA 3′ maturation. The formation of longer forms of the snoRNAs on depletion or mutation of snoRNP components is slightly surprising; if anything, the loss of these factors might have been expected to allow the exosome complex further access into the mature snoRNA region. This indicates that the snoRNP proteins play a more active role than simply blocking the exosome. They may be required to promote the final trimming reaction and/or to displace other factors that protect the 3′ ends of the pre-snoRNAs.
It was surprising that Nop1p was required for the accumulation of snoRNAs synthesized from polycistronic and intronic precursors but not monocistronic snoRNAs. These appear to have highly homologous box C+D regions and 3′-terminal stems, which likely constitute the snoRNP protein binding site. The obvious difference is that the monocistronic snoRNAs have a 5′-cap structure and therefore do not undergo 5′ processing by Rat1p. It is possible that in addition to influencing 3′ processing, Nop1p is also involved in protection of the 5′ ends during pre-snoRNA processing.
What is the function of Nop56p? Depletion of Nop56p inhibited cleavage at sites A0
, and A2
, leading to impaired synthesis of the 18S rRNA (Fig. ). These cleavages were also inhibited in strains with Nop1p, Nop58p, or the U3 and U14 snoRNAs depleted (17
), suggesting that Nop56p is required for the normal functioning of U3 and/or U14. Interestingly, the GAL::nop56
strains had distinct pre-rRNA-processing phenotypes. Greater inhibition of the cleavages at sites A0
, and A2
was seen on depletion of Nop56p than in the nop56-2
strain. In contrast, the nop56-2
strain had additional defects in the 25S rRNA synthesis pathway that were not observed on Nop56p depletion. The effects on ribosome synthesis of genetic depletion of Nop1p and conditional-lethal point mutations were also found to be distinctly different, and some mutations in NOP1
also interfere with 25S rRNA synthesis (42
In neither the strains with Nop56p depleted nor the nop56-2
strains does the reduction in the mature rRNAs appear sufficient to account for the severe growth inhibition. This is in contrast to depletion of Nop58p or Nop1p, both of which substantially reduced 18S rRNA levels. Ribose methylation of rRNA was also not greatly affected on Nop56p depletion. As previously proposed, Nop56p could be involved in the correct assembly of ribosomal particles (15
); mutations in NOP1
that lead to specific defects in ribosome assembly have been reported. Deletion of the cap binding complex proteins CBP20 and CBP80 (Gcr3p and Mud13p in yeast) was recently reported to be synthetic lethal with mutations in the snoRNP proteins Nop58p and Cbf5p (12
). In the absence of Gcr3p and Mud13p, some inhibition of pre-rRNA processing was observed, and synergistic defects in ribosome synthesis were proposed as the basis for the colethality (12
). Since the inhibition of rRNA synthesis does not appear to be the basis of the lethality seen on depletion of Nop56p, additional functions of the snoRNP proteins in other aspects of RNA metabolism appear possible. The cap binding complex is required for splicing commitment complex formation, and pre-mRNA splicing is therefore a possible target.
A homologue of Nop1p and a single homologue of Nop56p-Nop58p are present in Archaea
, where they may be associated with methylation guide RNAs (24
). We propose that Nop56p was derived from an ancestral Nop58p-like protein by gene duplication, but the proteins have clearly undergone substantial divergence in function.