Maturation of eukaryotic rRNAs is assisted by a large number of snoRNAs that either direct the posttranscriptional 2′-O-methylation and pseudouridylation of rRNAs or, less frequently, function in the nucleolytic processing of pre-rRNA. While the molecular function of modification guide snoRNAs is well understood, much less is known about the role that snoRNAs play in rRNA processing. The yeast snR30 is an essential box H/ACA snoRNA that is required for the nucleolytic processing of 35S pre-rRNA at the A0, A1, and A2 sites (36
). In this study, we have demonstrated that snR30 is an evolutionarily conserved snoRNA that is also present in all vertebrates, the fission yeast S. pombe
, and the unicellular ciliated protozoan T. thermophila
. More recently, we have identified a potential plant homologue of snR30 in Medicago truncatula
(our unpublished results), further supporting the notion that U17/snR30 is a ubiquitous snoRNA that is present in most, if not all, eukaryotic organisms.
Secondary structure modeling demonstrated that all U17/snR30 snoRNAs fold into the consensus hairpin-hinge-hairpin-tail structure of box H/ACA snoRNAs, except that they carry an additional internal hairpin located before the 3′-terminal hairpin of the RNA (Fig. , , and and data not shown). As indicated by deletion analysis of yeast snR30, neither the 5′-terminal nor the internal hairpin of U17/snR30 contains functionally important elements (Fig. ). Consistent with this notion, both the nucleotide compositions and sizes of these hairpins show a high level of variation during evolution. Expression of an snR5-snR30 chimeric RNA (R5-R30S) demonstrated that the 3′-terminal hairpin of snR30 carries all the elements that are critical for cell viability (Fig. ). The 3′ hairpin of each U17/snR30 RNA carries the conserved m1 (AUAUUCCUA) and m2 (AAACCAU) sequence elements, which occupy an invariant position in the proximal (lower) part of an internal loop, exactly 7 nucleotides upstream to the ACA box of the RNA (Fig. ). Mutational analyses demonstrated that the m1 and m2 motifs are crucial for cell viability and 18S production (Fig. and ). Apart from the essential m1 and m2 elements, the nucleotide sequence of the 3′-terminal hairpin of U17/snR30 RNAs shows no significant conservation, indicating that it contains no additional functionally important sequence elements.
One possibility is that the m1 and m2 motifs might function as specific protein-binding signals. Besides the Cbf5p/dyskerin (65 kDa), Gar1p (25 kDa), Nhp2p (22 kDa), and Nop10p (10 kDa) box H/ACA snoRNP core proteins (1
), purified yeast snR30 snoRNP was reported to be associated with three additional proteins of 36, 46, and 48 kDa (32
). However, in vitro reconstitution experiments performed with the human U17 snoRNA could detect only four U17-associated proteins, which, based on their apparent molecular masses, likely represent the human Cbf5p/dyskerin (60 kDa), Gar1p (29 kDa), Nhp2p (23 kDa), and Nop10p (14 kDa) box H/ACA core proteins (10
). Notably, in vitro reconstitution studies also revealed that the 3′-terminal hairpin of U17 could be packaged into a stable snoRNP particle (10
). In marked contrast, full-length snoRNAs were required for the assembly of the human U64 and U19 box H/ACA pseudouridylation guide snoRNPs. This observation strongly suggests that the 3′-terminal hairpin of U17 has specific structural and functional features which distinguish this RNA from the authentic pseudouridylation guide snoRNAs. Apparently, further experiments are required to clarify whether the m1 and m2 motifs of U17/snR30 can bind specific proteins.
The high degree of evolutionary conservation of the m1 and m2 motifs might also indicate that they represent antisense sequence recognition elements. In fact, the overwhelming majority of box H/ACA snoRNAs function as guide RNAs which select ribosomal pseudouridylation sites by forming transient base pairing interactions with complementary rRNA sequences. However, the fact that the m1 and m2 elements occupy the proximal part of the putative pseudouridylation loop of U17/snR30 rather than its distal part, as would have been expected for a genuine pseudouridylation guide RNA (14
), strongly argues against a modification guide function for U17/snR30. Moreover, human and yeast rRNAs lack sequences with known pseudouridylation sites that could form a canonical rRNA-guide RNA interaction with U17/snR30. At the moment, however, we cannot unambiguously rule out the formal possibility that U17/snR30 directs pseudouridylation of another, not yet identified RNA that plays a crucial role in pre-rRNA processing. Currently, we are testing whether the mouse U17 snoRNA is capable of directing pseudouridylation of appropriately designed artificial substrate RNAs expressed in the nucleoli of mouse cells.
According to another, more likely scenario, U17/snR30 might function as a molecular chaperone that facilitates the correct folding of pre-rRNAs, as has been proposed for the U3, U14, and U8 box C/D snoRNAs (see the introduction). Previous psoralen photo-cross-linking experiments found the human U17 (E1) and yeast snR30 snoRNAs to be associated with large pre-rRNAs (36
). Although vertebrate and yeast pre-rRNAs lack perfect, evolutionarily conserved complementarities to the m1 and m2 motifs of U17/snR30, numerous shorter, either perfect or imperfect base pairing interactions could be formed between the m1 and m2 motifs of U17/snR30 and pre-rRNA sequences (data not shown). This also implies that the m1 and m2 elements might form functionally important hydrogen bonding interactions with distinct regions of the pre-rRNA. Notably, alteration of the m1 or the m2 motif of snR30 gave rise to slightly different pre-rRNA processing pathways (Fig. ). In the absence of an intact m1 motif, processing of 35S pre-rRNA is delayed, while alteration of the m2 motif results in an increased accumulation of the aberrant 23S processing intermediate. Moreover, we have recently found that the viability of yeast cells expressing snR30m2, but not snR30m1, could be restored by coexpression of the human U17a snoRNA (our unpublished data). This observation, besides demonstrating that the human U17 snoRNA is indeed the functional homologue of yeast snR30, provides further support for the assumption that the m1 and m2 elements might work, at least to some extent, independently from one another. To identify the putative sites of interaction between U17/snR30 and pre-rRNA, we are currently performing a detailed cross-linking analyses of human and yeast pre-rRNAs.
Thus far, most box C/D and H/ACA snoRNAs implicated in rRNA processing have been assumed to be unique to either vertebrates (U8 and U22) or the yeast S. cerevisiae (snR30 and snR10). Only the U3 and U14 box C/D snoRNAs have been found in both yeast and metazoan cells. Demonstration that the U17/snR30 box H/ACA snoRNA is also present in vertebrates, the fission yeast S. pombe, and the protozoan T. thermophila further supports the idea that the snoRNA-assisted processing mechanism of eukaryotic pre-rRNAs has an ancient evolutionary origin. More importantly, determination of the functionally essential elements of U17/snR30 will greatly facilitate the future dissection of the molecular mechanism underlying the function of this snoRNA in pre-rRNA processing.