In this report we provide evidence that the essential protein YER036C/ARB1 in the GCN20 subfamily of ABC proteins is involved in the biogenesis of both 40S and 60S ribosomal subunits. Depletion of ARB1 in vivo led to a 30% to 40% reduction in the steady-state level of mature 18S rRNA and 40S subunits relative to 25S rRNA and 60S subunits. This deficit in 40S subunits can be attributed at least partly to a reduced rate of cleavage at the A0-A1-A2 sites in 35S pre-rRNA that produces the 20S precursor of 18S rRNA. Thus, depletion of ARB1 led to steady-state accumulation of 35S pre-rRNA and the aberrant 23S transcript that is generated by inappropriate cleavage at site A3 prior to A0-A1-A2 cleavage. We confirmed by pulse-chase analysis that the processing of 35S pre-RNA at the A0-A1-A2 sites is delayed in ARB1-depleted cells, and we also detected a diminished rate of 20S to 18S conversion in the same cells. We presume that both of these processing defects contribute to the overall ~40% reduction in the synthesis of mature 18S seen in ARB1-depleted cells, but it is difficult to assess their relative contributions to the defect in 40S biogenesis.
Apart from these defects in the rRNA processing reactions that produce 18S rRNA, we observed accumulation of the RPS2-GFP fusion protein in nuclear foci (one per cell) in roughly one-third of the ARB1-depleted cells, suggesting a partial defect in nuclear export of pre-40S particles. It is unclear whether ARB1 functions directly to stimulate 40S export or whether the apparent delay in export is an indirect consequence of the accumulation of aberrant pre-40S complexes in ARB1-depleted cells that are incompetent for nuclear export. As 20S to 18S rRNA processing clearly continues, albeit at reduced rates, in ARB1-depleted cells (Fig. ), the proposed defect in nuclear export of pre-40S particles would amount to a delay rather than a tight block of pre-40S export from the nucleus. However, such a delay in nuclear export could be responsible for the reduced rate of 20S to 18S rRNA processing detected in the pulse-chase analysis shown in Fig. . ARB1 could also enhance 20S to 18S conversion more directly by associating with pre-40S particles in the cytoplasm, as ARB1 itself is exported to the cytoplasm (Fig. ). In any event, it seems clear that ARB1 stimulates multiple steps of the 40S biogenesis pathway.
The A0-A1-A2 processing events occur in the 90S pre-ribosomal particle. The fact that the majority of ARB1 in cell extracts cosediments through sucrose gradients with IMP4 at a position corresponding to ~90S particles (Fig. ) is consistent with the possibility that ARB1 directly stimulates these cleavage reactions in the 90S preribosome. We also obtained evidence consistent with the possibility that ARB1 functions directly in pre-40S particles to stimulate nuclear export or to enhance 20S to 18S processing in the cytoplasm. Thus, we showed that ARB1 shuttles from nucleus to cytoplasm, and we observed that a fraction of ARB1 in cell extracts cosediments with 40S subunits. In addition, we found that ARB1 is weakly associated with DED1 and SCP160, both of which copurify with processing factors present in pre-40S particles (62
) and with 20S pre-rRNA (for DED1) (76
). If ARB1 is associated with 90S pre-ribosomes, as suggested above, it could remain bound to the pre-40S particle after cleavage of 35S pre-rRNA at the A0-A1-A2 sites and subsequently accompany the pre-40S to the cytoplasm. Alternatively, ARB1 could interact transiently with 90S and 40S preribosomal particles at various points along the processing pathway. The latter possibility is more consistent with the fact that ARB1 was not identified previously as a stable constituent of any preribosomal particles.
It is intriguing that depletion of ARB1 also produced a delay in processing of 27S pre-rRNA to 25S rRNA, leading to steady-state accumulation of 27S precursors. Accumulation of the 7S precursor to 5.8S rRNA was also evident in cells lacking ARB1. However, we did not observe a defect in nuclear export of GFP-RPL25 nor reduced steady-state levels of either 25S rRNA or 60S subunits in ARB1-depleted cells. Thus, while ARB1 is required for a WT rate of 60S biogenesis, it seems that all of the 27S pre-rRNA is eventually converted to 25S rRNA so that a normal steady-state level of 60S subunits is achieved in ARB1-depleted cells.
Several observations support the possibility that ARB1 also functions directly in pre-60S particles to stimulate processing of 27S and 7S pre-rRNAs. First, we found that a fraction of ARB1 cosediments with 60S ribosomal particles. Second, ARB1 is tightly associated with TIF6 and LSG1 and weakly associated with ARX1, all of which interact with pre-60S subunits and stimulate different aspects of 60S biogenesis. It is interesting that LSG1 appears to interact with pre-60S subunits only in the cytoplasm (30
), whereas ARX1 resides in nuclear pre-60S particles at a stage just prior to nuclear export (52
). Although TIF6 shuttles to the cytoplasm (65
), its function is also required further upstream in the 60S biogenesis pathway for efficient conversion of 27SB to 25S rRNA. Based on its physical association with these three factors, it is possible that ARB1 interacts with pre-60S subunits in both the nucleus and cytoplasm and, similar to TIF6 (65
), may accompany pre-60S subunits during export from the nucleus.
Most ribosome biogenesis factors present in the 90S preribosome are not stable constituents of 60S or 40S preribosomal particles, and many factors join the pre-40S or pre-60S particles after cleavage of 35S pre-rRNA in the 90S particle. Thus, the majority of biogenesis factors seem to function exclusively in pre-40S or pre-60S particles (24
). ARB1 belongs to a small group of factors that stimulate steps in both the pre-40S and pre-60S pathways, as well as the initial cleavage of 35S rRNA in the 90S particle. Interestingly, DED1, which interacts with ARB1, is associated with both pre-40S and pre-60S particles. It is also intriguing that RLI1, another soluble ABC protein, is a shuttling factor that interacts with pre-40S and pre-60S particles, enhances pre-rRNA processing reactions in both pathways, and stimulates export of both pre-40S and pre-60S particles (37
A number of GTPases and AAA-type ATPases have been implicated in 60S processing, but only the GTPase BMS1 (20
) and the ABC ATPase RLI1 (37
) have been identified in the 40S processing pathway (20
). Our genetic analysis indicates that the ATPase activity of ARB1 is required for its essential function in ribosome biogenesis. By analogy with other ABC proteins, and RLI1 in particular (35
), it can be predicted that binding of ATP will tightly clamp together the ABCs in ARB1, whereas ATP hydrolysis will lead to partial dissociation of the two cassettes. The opening and closing of this clamp could produce conformational changes in segments of the preribosomes or in ribosome-associated factors that stimulate rRNA processing or subunit assembly reactions. Given that ribosomal subunit export is energy dependent (33
), ATP hydrolysis by ARB1 might also perform work connected with the nuclear export of 40S preribosomes.
All of the previously characterized soluble ABC proteins in the GCN20 subfamily were shown to interact with ribosomes and to function in translation initiation (RLI1 and ABC50), translation elongation (YEF3), translational control (GCN20), or ribosome biogenesis (RLI1). Our findings allow us to extend this generalization to ARB1. Moreover, preliminary results indicate that the last uncharacterized member of this subfamily in yeast, NEW1, is also involved in ribosome biogenesis (J. Dong and A. G. Hinnebusch, unpublished observations). It will be interesting to identify the molecular features of the members of this subfamily of ABC proteins that dictate their interactions with ribosomes and identify the nature of the mechanical work they carry out to promote ribosome biogenesis or protein synthesis.