Our knowledge of ribosome synthesis in eukaryotes is based largely on studies performed in the yeast S. cerevisiae
. The insights gleaned from this organism are usually applicable to a wide range of eukaryotic species due to high conservation of ribosome components, homology between many assembly factors, and the overall similarity of pre-rRNA processing pathways. In some instances, however, pre-rRNA processing steps differ in lower and higher eukaryotes. One of the better-known examples is the use of additional snoRNAs, such as U8 and U22, in pre-rRNA processing in vertebrates (44
). Because of the relative paucity of data for higher eukaryotes, the molecular machinery that underlies the differences in ribosome maturation pathways is still very poorly understood. In this study, we characterized the novel DEAD box protein Ddx51, which plays a role in ribosome maturation in mammalian cells and functions in a pre-rRNA processing step that is mechanistically distinct from the corresponding step in yeast ribosome biogenesis. The evidence obtained by knocking down endogenous Ddx51 in mouse cells and expression of its dominant negative mutant is consistent with this factor playing a role in processing the 3′ end of 28S rRNA. Moreover, we found that expression of the Ddx51 mutant predicted to lack catalytic activity significantly increases the amount of U8 snoRNA base paired with pre-rRNA within preribosomal RNPs, suggesting that Ddx51 acts to promote U8 release during preribosome assembly.
Proteins of the DEAD box and related families of RNA helicases are found from bacteria to humans and participate in a multitude of cellular processes including translation initiation, pre-mRNA splicing, ribosome biogenesis, and RNA degradation (reviewed in references 6
, and 10
). All these proteins contain several conserved motifs involved in binding and hydrolysis of nucleoside triphosphates and are predicted to function by promoting rearrangements of RNA molecules and RNA-protein complexes (25
). A systematic analysis of the putative helicases in S. cerevisiae
showed that at least 20 of them are involved in ribosome biogenesis (5
). Several of these proteins have been implicated in facilitating the binding and release of snoRNAs from pre-rRNA: Dbp4p is required for the release of U14 (32
), Rok1 promotes the release of snR30 (7
), and Has1 is required for U3 and U14 snoRNA release (38
). Our analysis of Ddx51 indicates that this previously uncharacterized member of the family also acts by potentiating snoRNA release from preribosomes. Curiously, the target of Ddx51 is U8, a snoRNA found in higher eukaryotes but absent in S. cerevisiae
, implying that Ddx51 has no functional counterpart in budding yeast. Consistent with this notion, we find no significant similarity between the N-terminal region in Ddx51, which excludes the conserved helicase core, and those of any of the DEAD box helicases encoded in the yeast genome (data not shown). Thus, it is possible that the function of Ddx51 has evolved in higher eukaryotes to specifically facilitate the U8 snoRNA-dependent step in the biogenesis of their ribosomes.
A deficiency in Ddx51 function in mammalian ribosome maturation is manifested primarily as the inhibition of pre-rRNA cleavage at site 6, separating 28S rRNA and the 3′ETS region (Fig. ). The elevated levels of 46S pre-rRNA in preribosomes (Fig. ) also indicate that cleavages separating the 18S and 28S branches of the pathway (Fig. ) are slowed down, but it is unclear whether this is simply due to inefficient early assembly in general or whether the cleavages in ITS1 and 3′ETS are partially coupled in mouse cells, similar to the situation in yeast (1
). Overall, defects caused by mutant Ddx51 show a striking resemblance to the depletion of U8 snoRNA in the Xenopus
system: an accumulation of abnormal 28S precursors extended at the 3′ end, inhibition of the formation of mature 5.8S and 28S rRNAs, and decreased levels of 18S due to partial inhibition of processing in ITS1 (43
According to the existing model, U8 snoRNA binds to newly transcribed pre-rRNA and facilitates its proper folding but later needs to be displaced for the 3′ cleavage of 28S and downstream processing to occur (43
). Our study indicates that dissociation of U8 from pre-rRNA is not spontaneous but requires, or is enhanced by, Ddx51. The S403L mutation in the conserved helicase motif III in Ddx51 traps U8 in the pre-rRNA-bound state (Fig. ) and leads to the accumulation of particles containing pre-rRNAs that are internally cleaved in ITS1 but still possess unprocessed 3′ETS tails (Fig. ). These data are most easily interpreted as Ddx51 acting directly on U8, although we cannot exclude the possibility that Ddx51 might affect some other structural rearrangement in the preribosome particle, which causes U8 displacement indirectly. The same caveat applies to a number of other putative helicases proposed to act in snoRNA release and underscores the importance of developing methods to map intermolecular interactions within preribosomes with high precision.
Consistent with its having a role in ribosome biogenesis, Ddx51 is predominantly nucleolar (Fig. ) and cosediments with high-molecular-weight RNPs containing ribosome synthesis factors (Fig. ). In addition, we observe tight association of Ddx51 with salt-resistant core preribosomes (Fig. ), suggesting that the protein is stably integrated into these complexes. Unfortunately, we were unable to address the association with preribosomes in this study more directly because efficient immunoprecipitation of Myc-Ddx51 was found to require partially denaturing conditions, which disrupted its interactions (L. Srivastava and D. G. Pestov, unpublished observations). This is reminiscent of mammalian Nog1, which is also refractory to immunoprecipitation when it is incorporated into preribosomes (36
), suggesting that Ddx51 might be similarly buried inside these large RNPs, which limits epitope accessibility on this protein.
The interaction of Ddx51 with the preribosome-associated G protein Nog1 in vitro
and in the two-hybrid system (Fig. ) points to their cooperation in mammalian ribosome maturation, further supported by the overlapping processing defects caused by their depletion and dominant-inhibitory mutants (Fig. , ). Nog1 is a conserved protein essential for the maturation of 60S ribosomal subunits and is found in a broad range of pre-60S complexes in S. cerevisiae
), but the exact role of this protein is still enigmatic. In our previous studies, we found that a mutation predicted to restrict conformational flexibility in Nog1 caused multiple defects in ribosome maturation including blocked cleavages in ITS2, accumulation of stalled 32S pre-rRNA-containing complexes, and inhibition of 3′ETS processing (36
). Similar defects were also observed with certain mutations in the pre-60S ribosome assembly factors Bop1 and Pes1 (35
). Our results from this study suggest a possible explanation for the linkage between pre-60S ribosome factors and 3′ETS processing. In particular, the finding that U8 dissociation from pre-rRNA is inhibited by mutant Nog1 raises the possibility that U8 release might depend on the assembly of the proper pre-60S processing complex on a nascent transcript. Since preribosomes containing Nog1G224A are misassembled and unable to progress through maturation, as evidenced by the accumulation of stalled particles in fraction 5 (Fig. , right), they might be in the wrong conformation to stimulate the Ddx51 activity required for effective U8 release. In this model, Ddx51 would not only provide mechanical assistance to displace U8 but also serve as part of a checkpoint enforcing the optimal order of events during ribosome assembly.