Protein-based tandem affinity purification of yeast pre-ribosomal particles at different stages of maturation identified a tremendous number of novel biogenesis factors and revealed valuable details of the yeast ribosomal assembly pathway (2
). In the hope of identifying ribosomal processing factors which directly interact with the yeast snR30 snoRNP, we established an entirely RNA tag-based tandem affinity purification procedure to purify snR30 under natural conditions (). The principle of our approach was based on a previously reported RNA affinity purification protocol developed for isolation of mammalian 7SK snRNP (39
). However, instead of applying one PP7 coat protein- and a short tobramycin-binding RNA motif (39
), we fused three MS2 coat protein-binding sites and an extended version of the tobramycin-binding motif to the 5′
end of snR30 to increase the specificity and efficiency of the affinity selection reactions (33
). Upon expression in yeast cells, the inserted tag sequences folded correctly and independently from snR30, because they did not interfere with the accumulation and function of the tagged snR30 and more tellingly, they proved to be fully active in affinity selection. We propose that upon adopting appropriate RNA expression strategies, the RNA-based tandem affinity purification technique described here may be a powerful tool to define the protein composition of in vivo
assembled RNPs in various organisms.
Affinity purification of yeast snR30 followed by co-IP and cross-linking experiments demonstrated that besides the four H/ACA core proteins, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. It remains unclear whether Utp23p and Kri1p directly recognize the snR30 snoRNA or they interact with the snR30-associated snoRNP proteins. Genetic depletion of Utp23p and Kri1p had no effect on snR30 accumulation, excluding the possibility that these proteins function in the biogenesis of the snR30 snoRNP. Both Utp23p and Kri1p had been reported to be integral components of the small subunit processome and demonstrated to be essential for 35S pre-rRNA processing at the A0
). Since Utp23p and Kri1p interact with snR30 in a mutually exclusive manner, they likely participate in distinct steps of snR30 function (C). Consistent with this idea, depletion of Utp23p, but not Kri1p, dramatically increased the accumulation of snR30 in large pre-ribosomal complexes, indicating that Utp23p promotes the pre-ribosomal release of snR30 (C). Utp23p features a putative PIN (Pi
terminus) domain that is presumed to confer nuclease activity to a group of proteins participating in RNA metabolism (40
). However, alteration of the PIN-like domain of Utp23p had no effect on cell viability, making it unlikely that Utp23p functions as a ribonuclease in 18S rRNA production (27
). Depletion of Utp23p had no influence on the preribosomal association of other processing snoRNAs, underlying its specific functional interaction with snR30. It remains unclear how Utp23p can promote the pre-ribosomal release of snR30. It is unlikely that Utp23p works together with the Rok1p RNA helicase that seems to be responsible for specific unwinding of snR30 and 35S pre-rRNA (37
), since Rok1p associates with the 35S pre-rRNA and seems to disrupt its base-pairing interaction with snR30 even in the absence of Utp23p (). On the other hand, it is also conceivable that snR30, although recruited to pre-ribosomal particles, is unable to base-pair with 35S sequences without Utp23p. Nevertheless, the lack of snR30-35S base-pairing interaction in preribosomes devoid of Utp23p indicates that the anomalous retention of snR30 is mediated by stable association with pre-ribosomal protein(s) which probably interact transiently with snR30 under normal conditions. Therefore, affinity purification of snR30 from Utp23p-depleted cells may identify the putative snR30 retention factors which likely represent ribosomal processing factors directly collaborating with snR30.
Co-transcriptional assembly of the small subunit processome on the nascent 35S pre-rRNA is believed to commence with recruitment of the preassembled UTP-A processing complex (43–46
). Early binding of UTP-A is essential for stepwise incorporation of other modules of the 90S processome, including the UTP-B and UTP-C protein complexes and the U3 snoRNP. Proper incorporation of Utp23p and Kri1p into pre-ribosomes or in other words, assembly of functionally active small subunit processome depends on the presence of snR30 () (26
). Given that snR30 can bind to pre-ribosomes independently of Kri1p and Utp23p, preribosomal docking of these proteins is likely preceded by recruitment of snR30. Of course, we cannot exclude the possibility that one of these proteins, either Utp23p or Kri1p, is recruited to the small subunit processome together with snR30 as a preassembled snoRNP complex. Nevertheless, we favour the idea that Utp23p and Kri1p interact with snR30 only within the newly assembled pre-ribosomal particle. Interestingly, recruitment of snR30 and Utp23p to the nascent 35S pre-rRNA does not depend on the pre-incorporation of the UTP-A early processing complex (Supplementary Figure S1
). This might indicate that snR30 and Utp23p play a very important role in the assembly, dynamics or function of the small subunit processome.
During preparation of this article, yeast snR30 has been reported to associate with the nucleolar protein Nop6, the small ribosomal subunit proteins S9 and S18 and histones H2B and H4 (26
). However, as acknowledged by the authors, none of these proteins are specific for snR30, since they also interact with other partially overlapping sets of both H/ACA and C/D snoRNAs (26
). Thus, similarly to the previously proposed putative snR30-associated proteins (20
), these new snR30 proteins likely bind to snR30 indirectly, as common components of large complexes. Importantly, none of the earlier detected snR30 proteins were present in our snR30 preparation, most probably because in our purification procedure, but not in the protocols used by others, large complexes were eliminated from the extract by extensive ultracentrifugation prior to snR30 selection.
In summary, by employing a novel RNA affinity purification protocol, we demonstrated that the Utp23p and Kri1p small subunit processing factors specifically and transiently interact with the yeast snR30 snoRNP. Utp23p may promote structural rearrangements of the pre-ribosome, essential for snR30 release. We also showed that Utp23p binds to the nascent pre-ribosome independently of the early-binding UTP-A complex, raising the possibility that it plays a more central role in ribosome biogenesis than anticipated before.