The U3 snoRNA is an exceptional snoRNA in that its base pairing with the pre-18S rRNA leads to pre-rRNA cleavage events. We have asked whether the U3 snoRNA plays a role in the pre-rRNA structural changes in the 5′-ETS and in the pre-18S rRNA sequences that participate in the 5′-end pseudoknot (nucleotides 1–220; 980-1150). Using in vivo chemical probing, we demonstrate that the structure of these pre-ribosomal sequences depends on the presence of the U3 snoRNA, and thus provide direct evidence for a function in pre-18S folding for the U3 snoRNP and its associated proteins. Furthermore, in vivo chemical probing, mutational analysis and genetic ‘rescue’ revealed a third previously unexplored (in yeast) U3 snoRNA:pre-rRNA base pairing interaction where the 3′-hinge at nucleotides 62–72 in the U3 snoRNA is base paired to nucleotides 281–291 in the 5′-ETS. This U3 snoRNA:5′-ETS interaction is a prerequisite for the earlier reported interaction between the 5′-hinge of the U3 snoRNA and nucleotides 470–479. We show that the 3′-hinge interaction of the U3 snoRNA with the 5′-ETS is essential for growth and required for the subsequent binding of other SSU processome proteins.
These results suggest that, temporally, the 3′-hinge base pairing with the 5′-ETS is likely the first RNA:RNA interaction to occur between the U3 snoRNP and the pre-rRNA. The 5′-ETS sequence that base pairs to the 3′-hinge is the first among the base pairing sequences to be transcribed by RNA polymerase I. Furthermore, while the 3′-hinge interaction is required for the 5′-hinge interaction to occur, the reverse is not true.
Our results are in agreement with previous chemical probing studies of the yeast U3 snoRNA in an RNP in vivo
and in vitro
). Analysis by chemical probing of U3 snoRNA sequences confirmed the base pairing of the 5′-hinge and of Box A, Box A′ and the GAC Box to the pre-rRNA. Interestingly, because of the lack of nucleotide reactivity to DMS in vivo
, Mereau et al.
) also proposed that nucleotides 60–64 of the 3′-hinge are base paired to the pre-rRNA. Additionally, they observed a decrease in reactivity of nucleotides 68–72 of the 3′-hinge in vivo
versus in vitro
, further supporting the hypothesis that these sequences are involved in base pairing. These studies were limited to probing the U3 snoRNA, so they did not query the base paired nucleotides in the pre-rRNA.
We have found that base pairing of the 3′-hinge sequence of the U3 snoRNA with the 5′-ETS and its essential function is conserved to budding yeast. This base pairing interaction has not previously been reported in S. cerevisiae
, though it had been previously described in both Xenopus laevis
and in Trypanosoma brucei
as essential for SSU biogenesis in these organisms (22
). Interestingly, the A′ cleavage site, which is U3-dependent, is upstream of the 3′-hinge base pairing site in both X. laevis
and T. brucei
. Since we have shown here that the 3′-hinge base pairs in yeast as well, it is also plausible that there is an A′ cleavage site associated with it. The presence of an A′ cleavage site in yeast would confirm that indeed the pre-rRNA cleavage events dependent on the U3 snoRNA are conserved among eukaryotes, since the U3 dependent cleavage sites (A′, A0
) have also been observed in mice (39
) and humans (40
). Furthermore, results from mutational analysis of the corresponding 3′-hinge of the human U3 snoRNA are also similar to those presented here (41
). Among the predicted three human U3 snoRNA base pairing sites, disruption of the 3′-hinge sequence reduced human Mpp10 co-immunoprecipitation to the greatest extent. Moreover, an intact 3′-hinge sequence was required for subnucleolar localization of the U3 snoRNA.
In vivo chemical probing is a powerful tool to monitor changes in RNA structure. The particular nucleotides examined by in vivo chemical probing are determined by the characteristic reactivity of bases to DMS and by the capacity of reverse transcriptase to stall at the modified base. However, not all nucleotides are equally reactive or accessible to DMS since they may be base paired or protected by proteins. For example, we can directly detect the 5′-hinge base pairing to the 5′-ETS (nucleotides 470–479) because many of the nucleotides involved are A’s and C’s whose modification is easily detected by primer extension without further manipulation. Nevertheless, it is not possible to directly ascertain the base pairing of the 3′-hinge of U3 snoRNA to nucleotides 281–291 of the 5′-ETS because these nucleotides are mainly G’s and U’s, and their modification is not detectable in this assay. Instead, we were able to detect a specific chemical modification pattern associated with the 3′-hinge base pairing interaction nearby between nucleotides 290–330. Thus, it is the combination of mutational analysis with in vivo DMS probing that has enabled us to delineate a new U3 snoRNA:5′-ETS base pairing interaction.
In contrast, the presence or absence of the U3 snoRNA does not influence the reactivity of the first 280 nucleotides of the 5′-ETS of the pre-rRNA. This suggests that the first 280 nucleotides of the 5′-ETS likely interact with other components of the SSU processome, before the first U3 snoRNA base pairing occurs at nucleotides 281–291. One strong candidate is the SSU processome subcomplex, UtpA/t-Utp, which is required for both optimal transcription of the rDNA and pre-rRNA processing (11
). The UtpA/t-Utp sub-complex interacts with the pre-rRNA even when U3 snoRNA is not expressed (11
). Furthermore, its assembly onto the pre-rRNA is independent of the presence of other SSU subcomplexes (12
) and prior binding of the UtpA/t-Utp subcomplex to the pre-rRNA is required for the subsequent assembly of other SSU processome subcomplexes (12
Using the 3′-hinge, 5′-hinge and Box A mutant U3 snoRNAs, we asked whether any or all of these base pairing interactions are necessary for SSU processome formation. To assay for SSU processome formation, we turned to co-immunoprecipitation of two proteins from different subcomplexes (Utp5 and Mpp10) and co-immunoprecipitation of the U3 snoRNA with Utp5. The Mpp10 protein was originally used as one of the ‘hooks’ to purify the SSU processome (8
), and its presence is required for SSU processome formation as detected in Miller spreads (42
). The UtpA/t-Utp subcomplex, of which Utp5 is a member, does not co-immunoprecipitate the U3 snoRNA except in the presence of the pre-rRNA (11
). Co-assembly of Utp5 with Mpp10, and co-immunoprecipitation of the U3 snoRNA with Utp5, are therefore both good indications of macromolecular assembly of the SSU processome. When we assay the effect of disruption of the U3 snoRNA base pairing interactions, we find that the 3′-hinge base pairing interaction with the pre-rRNA is most important for SSU processome formation, followed by the 5′-hinge interaction and then by the Box A interaction. Indeed, the 3′-hinge interaction is sufficient for some degree of SSU processome formation as a 5′-truncated U3 snoRNA that cannot base pair with either the 5′-hinge or Box A complementary sequences but can base pair with the 3′-hinge complementary sequence (-63) becomes incorporated into the SSU processome while a truncated form missing all three interactions (-72) does not.
Together with previously published findings, the results presented here provide a more detailed picture of the initial steps in SSU processome assembly. Assembly begins by protein subcomplex interactions with the nascent pre-rRNA, with the UtpA/t-Utp subcomplex likely the first to bind (A). Because the UtpA/t-Utp and UtpB subcomplexes co-immunoprecipitate in the absence of the U3 snoRNA (data not shown), the UtpB subcomplex is likely to assemble on the pre-rRNA following UtpA/t-Utp (13
) and prior to the U3 snoRNP. Other, yet to be identified, SSU processome proteins may also assemble at this point. Next, the U3 snoRNP assembles into the nascent SSU processome via base pairing to the pre-rRNA using its 3′-hinge sequence (B). This RNA:RNA interaction recruits the Mpp10 subcomplex (C). The two subsequent U3 snoRNA: pre-rRNA interactions occur, and other components likely assemble. The sequential binding at each U3 snoRNA base pairing site from 5′ to 3′ on the pre-rRNA is likely to be a signal for the assembly of other components of the SSU processome. Therefore the U3 snoRNP and its associated proteins coordinate, via U3 snoRNA base pairing, the pre-18S rRNA folding that leads to the cleavage events that release the mature 18S rRNA. These results thus support the proposal that the U3 snoRNP and its associated proteins play an important role in the folding of pre-18S rRNA.
Figure 9. Model for the initial assembly steps of the SSU processome. (A) The UtpA/t-Utp subcomplex is assembled first onto the nascent pre-rRNA, followed by the UtpB subcomplex and, probably, some other components of the SSU processome. (B) The U3 snoRNP anchors (more ...)