The yeast snR30 box H/ACA snoRNA has long been known to have an essential function in 18S rRNA processing, but the molecular mechanism supporting its function remained unknown (Bally et al, 1988
; Morrissey and Tollervey, 1993
; Atzorn et al, 2004
). In this study, by using a novel in vivo
crosslinking RNA affinity selection approach and yeast molecular genetics, we demonstrated that during pre-rRNA processing, two short internal sequence motifs of the 18S rRNA, called rm1 and rm2, transiently base-pair with the previously identified, functionally essential m1 and m2 sequences of snR30. We showed that formation of the newly discovered m1/rm1 and m2/rm2 Watson–Crick interactions of snR30 and 18S rRNA is required for 18S production and cell viability. The m1 and m2 motifs of snR30/U17 snoRNAs and the complementary rm1 and rm2 sequences of 18S/17S rRNAs are conserved in all vertebrates, budding and fission yeasts and in the unicellular protozoan T. thermophila.
Even more tellingly, the m1/rm1 base-pairing interaction of snR30 and 18S rRNA in T. brucei
is preserved by compensatory base changes. Thus, we can predict with great certainty that the snR30/U17 snoRNA has an evolutionarily conserved function in pre-rRNA processing.
Similarly to the antisense elements of H/ACA pseudouridylation guide RNAs, the m1 and m2 18S recognition motifs of snR30 are located on the opposite strands of an internal pseudouridylation loop-like structure of the 3′-terminal hairpin of the snoRNA. However, contrary to this obvious resemblance, there are fundamental structural differences between the interaction of pseudouridylation guide RNAs formed with their target sequences and the proposed association of snR30 with 18S rRNA. The antisense elements of H/ACA guide RNAs are located in the upper (distal) part of the pseudouridylation loop (), whereas the m1 and m2 elements of snR30/U17 snoRNAs occupy the lower (proximal) part of an internal loop of the 3′-hairpin (). The pseudouridylation guide elements bind to contiguous rRNA sequences, whereas the m1 and m2 elements interact with two distantly located sequence motifs that are folded together by an evolutionarily conserved stem-loop structure of 18S/17S rRNAs (Alkemar and Nygard, 2006
). Consequently, snR30/U17 forms a complex three-dimensional local structure with 18S/17S sequences that is novel to box H/ACA RNAs and according to our knowledge no similar interaction has been reported for other cellular RNAs either ().
Figure 7 Functionally essential elements of yeast snR30. The 5′-terminal and internal hairpins (shown schematically) lack functionally important elements (Atzorn et al, 2004). Sequences binding either box H/ACA or snR30-specific proteins are in shaded (more ...)
Determination of the crystal structure of archaeal H/ACA pseudouridylation guide RNP revealed a great structural flexibility for the pseudouridylation loop of substrate-free H/ACA RNA (Li and Ye, 2006
). Although the ACA box sequences, the lower and upper stem regions of the single-hairpin archaeal H/ACA RNA, are tightly fastened to the composite surface of the Cbf5, L7ae and Nop10 core H/ACA RNP proteins, the pseudouridylation loop nucleotides show very few intermolecular contacts with RNP proteins. This structural plasticity of the entire pseudouridylation loop indicates that both the distal and proximal loop nucleotides are available for base-pairing interactions and provides the structural basis for the interaction of the m1 and m2 proximal loop sequences of snR30 with pre-rRNA sequences. Identification of a novel target recognition strategy for the ‘pseudouridylation loop' of snR30 further emphasizes the functional diversity of H/ACA RNAs and in the future, it may facilitate understanding of the molecular role of additional ‘orphan' H/ACA RNAs.
Besides snR30, the nucleolytic processing of eukaryotic rRNAs also requires the U3, U8, U14 and U22 box C/D snoRNAs. However, these snoRNAs do not seem to be directly involved in nucleolytic pre-rRNA cleavage reactions. They are believed to function as RNA chaperones that transiently base-pair with pre-rRNA to safeguard its correct folding (Beltrame and Tollervey, 1995
; Liang and Fournier, 1995
; Hughes, 1996
; Peculis, 1997
; Sharma and Tollervey, 1999
; Borovjagin and Gerbi, 2000
). In principle, snR30 may also facilitate the proper temporal folding of the maturing pre-rRNA, as its binding prevents the formation of the internal stem loop that includes the rm1 and rm2 sequences in the mature 18S rRNA (Alkemar and Nygard, 2006
). However, we demonstrated that in addition to the m1 and m2 18S rRNA recognition elements, snR30 carries another functionally essential element located in the C561–G566/C572–A577 distal region of its 3′-hairpin (). The C561–G566/C572–A577 sequences lack obvious complementarity to pre-rRNA sequences and they are dispensable for interaction with 35S pre-rRNA (). Thus, we favour the idea that the distal part of the 3′-hairpin of snR30, instead of selecting pre-rRNA sequences, functions as a snoRNP protein-binding site. The terminal stem-loop regions of H/ACA RNA hairpins frequently carry docking sites for RNP proteins that target H/ACA scaRNPs to the Cajal body or control processing of the human telomerase RNA (Richard et al, 2003
; Jády et al, 2004
; Theimer et al, 2007
). Besides the four H/ACA core proteins, the yeast snR30 snoRNP has been reported to contain at least three snR30-specific snoRNP proteins of about 38, 46 and 48 kDa (Lübben et al, 1995
). We propose that binding of putative snoRNP proteins to the 3′-hairpin of snR30 is essential for the assembly of functional 90S pre-ribosomal particle and for processing of 18S rRNA (). The snR30 snoRNA, but not other H/ACA and C/D snoRNAs, is released from the maturing pre-rRNA by the Rok1 RNA helicase, suggesting that Rok1 either specifically recognizes the snR30 snoRNP or it is an integral component of this particle (Bohnsack et al, 2008
). Identification and functional characterization of the putative snR30-specific snoRNP proteins, which likely interact with the C561–G566/C572–A577 region of the 3′-hairpin of snR30, will be an exciting task for the future.
Thus far, all H/ACA RNAs assigned to a cellular function have been shown to function as guide RNAs that carry specific target recognition antisense elements to select substrate nucleic acids, such as rRNAs (snoRNAs), snRNAs (scaRNAs) or telomeric DNA (telomerase RNA). Other parts of the H/ACA guide RNAs tether the Cbf5/dyskerin pseudouridine synthase or the telomerase reverse transcriptase to the selected target nucleic acid. In this study, demonstration that the m1 and m2 sequences of snR30 function as 18S recognition elements, which probably target essential snR30-associated processing factors to pre-rRNA, indicates that not only pseudouridylation guide RNAs and telomerase RNA but also snR30 functions as a guide RNA.