By using a strain in which Cbf5, the Ψ synthase enzyme associated with the box H/ACA snoRNP complexes, is compromised for activity, we have shown that while the formation of the Ψ in S. cerevisiae
5.8S rRNA is associated with snoRNP activity, the formation of the Ψ in S. cerevisiae
5S rRNA is independent of Cbf5. Our analysis suggests that 5.8S rRNA modification at this position is not unique to S. cerevisiae
and most likely occurs in a wide range of hemiascomycetes, a group that covers an evolutionary breadth comparable to that of the entire chordate phylum (24
). Likewise, the formation of the Ψ in 5S rRNA has been reported for other hemiascomycetes (75
The synthesis of 5.8S rRNA in yeast is complex (27
), and we have now shown that an snoRNA is involved; snR43 uses the same guide sequences that function elsewhere (position 966 in 25S rRNA) to direct the pseudouridylation of 5.8S rRNA. snR43 adds to the list of yeast snoRNAs that guide pseudouridylation at more than one site in rRNA, bringing the total to 13, nearly half of the 28 guide H/ACA snoRNAs. snR43 and snR49 are the only S. cerevisiae
snoRNAs known to use the same guide domain to target the pseudouridylation of two different species of mature rRNA.
The nucleotide equivalent to yeast 25S-Ψ966 in human 28S rRNA also is a Ψ (83
), and the site is one of two assigned to the 133-nt snoRNA ACA9 (54
). Only the guide elements in ACA9 show any resemblance to snR43, constrained by the need to base pair with a homologous site in 28S rRNA; ACA9 does not target human 5.8S rRNA. A similar phenomenon has been noted for the yeast and human snoRNAs guiding the highly conserved Ψs in helix 69 of large-subunit rRNA (4
). However, in that case the overall secondary structure and length both are similar, unlike the case of yeast snR43 and its human counterpart, for which even those characteristics differ markedly due to the insert stem-loop and the greater than 70-nt difference in size. Human and plant 5.8S rRNAs do not contain a Ψ at a position equivalent to that of yeast 5.8S-Ψ73, yet neighboring nucleotides are modified (Fig. ) and assigned to particular snoRNAs (14
Given that guide H/ACA snoRNAs are involved in the formation of Ψs in transcripts produced by polymerases other than RNAP I, the fact that no snoRNA is involved in the modification of 5S rRNA, a product of RNAP III, was not obvious a priori. By testing known RNA guide-independent Ψ synthases of yeast, we have identified Pus7 as the synthase catalyzing the formation of the Ψ in 5S rRNA. This is the first report of a Ψ in cytoplasmic eukaryotic rRNA that is not snoRNA guided. Importantly, now all 46 U's known to be converted to Ψs in S. cerevisiae rRNA are associated with a modifying trans-acting factor.
On the basis of transcriptional coregulation, Pus7 was included in a set of genes that function in ribosome and rRNA biosynthesis (109
). Here, we have directly linked Pus7 activity to the modification of 5S rRNA. The targets of Pus7 all share a 7-nt consensus at the site of modification (9
). How Pus7 recognizes its natural targets still is not known. Presumably, there is more determining a Pus7 substrate than the short 7-nt consensus encompassing the modification site, because matches are moderately frequent in RNA (data not shown). Moreover, even within a recognized substrate, only particular sites are modified, while other matches are not. Particularly illuminating is the fact that roughly 30 nt downstream of the site pseudouridylated by Pus7 in S. cerevisiae
5S rRNA (nt 79 to 85) is a perfect match to the particular version of the 7-nt sequence found at the site of Pus7 action in U2 snRNA, yet position 50 is the only Ψ in S. cerevisiae
5S rRNA (75
). Similarly, U2 snRNA has a match downstream (nt 52 to 58) as well, within a region previously shown to be important for modification by Pus7, yet this site is not modified either (62
). The inability of Pus7 to modify substrate RNAs at additional sites that match the consensus may be due to the masking of these sites by a protein(s). Alternatively, only certain portions of substrates may be suitable for modification by Pus7, and perhaps only at certain stages of RNA folding, as seen with other Ψ synthases and enzymes modifying uridine (41
). Consistent with this possibility, only an altered conformation of tRNA, called the lambda form (42
) and featuring an unfolded D-stem/loop, could be docked computationally into the presumed active site of E. coli
). Until now, natural substrates for studying Pus7 have been in short supply; our discovery that the very abundant 5S rRNA is a Pus7 substrate means that the pus7
Δ strain should provide large amounts of 5S rRNA/RNP substrate, thereby leading to a better understanding of recognition and catalysis by Pus7.
Until recently, the prevailing view was that the pseudouridylation and 2′-O methylation of eukaryotic cytoplasmic rRNA were guided by snoRNAs in snoRNP complexes. Now, for each of the two classes of nucleotide modification that are introduced primarily by snoRNPs, an exception has been identified in which rRNA modification depends on classic protein-only enzymes. Our elucidation of Pus7 as the synthase responsible for the pseudouridylation of 5S rRNA, combined with the revelation that the protein Spb1 acts in the 2′-O methylation of a site in yeast 25S rRNA (58
), means that snoRNP-independent mechanisms need to be considered for both types of rRNA modification. The reason for the different mechanisms and particular targets is not obvious. Possible reasons for different mechanisms are that (i) separate machinery enables coupling to separate regulatory networks, (ii) they assure that certain modifications are formed irrespective of the bulk of rRNA modifications, and (iii) the bacterium-like site-specific protein mechanism may have been retained from a common ancestor (Spb1 is related to RrmJ, the enzyme that modifies the neighboring site in bacteria) (58
). While there presently is a paucity of knowledge on their exact role, no single snoRNP-catalyzed modification has been identified as being overly critical in yeast (8
). Perhaps the rRNA modifications introduced by guide RNA-independent enzymes are more important and necessitate separate machinery. Whereas the Spb1-guided modification has been shown to be essential for normal ribosome production and translation (58
), the significance of the Pus7-catalyzed Ψ in 5S rRNA is not obvious from its position, and a critical role is not supported by phylogenetic conservation outside of ascomycetes.
The pseudouridylations of the RNAP II-transcribed U2 snRNA and the RNAP I-transcribed pre-rRNA are known to be linked by an snoRNP-dependent pathway; snR81 uses different guide domains to target a Ψ site in rRNA and one in U2 snRNA (62
). We have now demonstrated a link between the pseudouridylation of U2 snRNA and the RNAP III-transcribed 5S rRNA by the snoRNA-independent synthase Pus7, which also participates in the modification of tRNAs (RNAP III transcripts). The 5S rRNA genes are interspersed between the rRNA repeats in S. cerevisiae
and are, by definition, nucleolar. The observed spatial juxtaposition of pre-tRNAs and the nucleolus indicates that a major part of tRNA biogenesis also is compartmentalized in the nucleolus with rRNA synthesis and ribosome assembly (11
). In fact, additional experiments have shown that tRNA genes are proximal to the nucleolus in yeast (34
). Finally, snRNAs have been detected in the Xenopus laevis
nucleolus, in which some of the modifications occur (57
). These observed associations suggest the possibility that Pus7 activity is connected to the nucleolus and, more importantly, point to the possibility that the modification status provides a signal allowing the coregulation of the production and turnover of rRNAs, spliceosomes, and tRNAs. These observations also suggest a link to the production of ribosomal proteins, since the Pus7-catalyzed Ψ35 in U2 snRNA is important for the high-efficiency splicing of S. cerevisiae
), which are preferentially located in ribosomal protein genes (32
). Indeed, the interdependencies are consistent with the concept that the production of RNAP II and RNAP III transcripts is regulated in response to pre-rRNA synthesis via an extensive network of interactions (18
Recent work has shown that the modification of tRNA is important for its stability and that specific degradation pathways are triggered by undermodification (1
); thus, the hypomodification-triggered degradation of RNA could provide a means of coupling modification activity to a regulatory network. Interestingly, Pus7 is one of several factors directly linked to a rapid tRNA degradation pathway that acts on specific hypomodified tRNAs (1
). Another RNA degradation pathway triggered by tRNA hypomodification also shows links to 5S rRNA synthesis in yeast (48
). Furthermore, the interaction of 5S rRNA and ribosomal protein L5 is critical for 5S rRNA stability and incorporation into the pre-60S ribosomal particle (22
), and it has been proposed that the unstable nature of unbound 5S rRNA probably is connected to a lack of modification and that this is part of a network keeping pre-rRNA processing linked to RNA polymerase III activity (13
). Interestingly, altered 5S rRNA stability also is observed when RNAP I activity is uncoupled genetically from attenuating its activity in response to stress (18
). Our elucidation of Pus7 as the factor involved in the pseudouridylation of yeast 5S rRNA will facilitate the further examination of the possible link between 5S rRNA stability and modification.
The specificity for multiple sites demonstrated by the factors studied here has important implications for assigning functions to enzymes or guide RNAs in other organisms. Pus7 shows multisite, multisubstrate specificity that is unparalleled. Furthermore, snR43 shows dual specificity; the same guide elements of snR43 are used to modify pre-rRNA at different sites that correspond to mature 5.8S and 25S rRNA sequences. The exact base-pairing potential seems to differ slightly, with the pairings for the 5.8S rRNA target being lengthier (Fig. ). The high degree of flexibility in the guide pairings (Fig. ) makes the assigning of function difficult for organisms in which the exact modifications have not been mapped or in which the stringent verification of the guide RNA by effecting the disruption of its activity is not convenient. Likewise, the increasing number of targets for bacterium-like Ψ synthases makes assigning the exact extent of their functions more tentative. Moreover, for enzymes or guide RNAs for which the disruption of activities causes observed phenotypes, one has to be cautious in assigning roles to particular modifications when unidentified ones may yet exist.