In this study, we have found that a single substitution of alanine for a conserved glycine in the G domain of Nog1 produces a remarkably strong dominant-inhibitory effect that virtually shuts down the biosynthesis of 60S ribosomal subunits in mammalian cells. The G224A mutant of mouse Nog1 binds GTP, localizes to the nucleolus and readily associates with ribosome precursor particles like the wild-type protein. The presence of the mutant protein, however, causes severe perturbations of the entire 60S assembly process by stalling the maturation of nucleolar pre-60S particles and inducing aberrant processing and degradation of the newly synthesized 5.8S/28S rRNA precursors. Because dominant-negative phenotypes have not been observed previously for Obg proteins, the mutation described here introduces a useful new tool for the investigation of molecular mechanisms in this family of GTPases.
Our results demonstrate that mammalian Nog1, like its orthologs from lower eukaryotes, is required for ribosome synthesis. Despite the identification of multiple preribosome components associated with Nog1p in yeast (11
), the exact role of this protein in ribosome assembly is not known. Our data show that the impairment of a critical pivot point in the G domain of Nog1 allows for the assembly of preribosomal complexes but creates particles from which bound components apparently fail to disengage. In particular, the dramatic changes in the amount and properties of nucleolar preribosomes occurring after G224A expression (Fig. and ) indicate that Nog1 function is required for the productive maturation of complexes containing 32S pre-rRNA. It is possible that the deficiency of factors due to sequestration by the stalled 32S-containing precursor complexes is also the primary reason for the multiple anomalies in pre-rRNA processing observed in cells expressing this mutant, ultimately leading to the degradation of the newly synthesized 28S/5.8S pre-rRNA (Fig. and ).
The assembly of each ribosomal subunit in vivo involves the binding and dissociation of dozens of processing factors. How these binding/dissociation events are controlled mechanistically is not well understood. Recently, studies of yeast have implicated two cytoplasmic GTPases, Efl1p/Ria1p and Lsg1p, in facilitating the release of the preribosome-associated shuttling proteins Tif6p and Nmd3p from nascent subunits in the cytoplasm (13
). Our data raise an interesting question of whether Nog1 might similarly mediate the release of specific factors bound to preribosomes, but during their earlier, intranucleolar maturation steps. Although we do not yet understand the mammalian ribosome synthesis machinery well enough to determine the identity of factors affected by Nog1 function, this issue may be well worth addressing in better explored systems.
The critical role of the glycine 224 residue in Nog1 suggests that conformational rearrangements of the switch II element are the key factors in Nog1 activity after it binds to pre-60S ribosomes. The essential role of the pivotal glycine residue within the DXXG motif for the mobility of the switch II element is well established, explaining the universal conservation of this residue in GTPases (1
). Replacement of the glycine with any other residue impairs conformational changes and results in altered protein interactions in a number of GTPases, including Ras, Gαs
, and EF-Tu (19
). The crystal structure of Ras with the corresponding G60A mutation has been solved recently (8
). This study showed that the substitution of the switch II glycine created a novel open conformation in the G domain that precluded the binding of effectors but at the same time stabilized bound Sos, a guanine nucleotide exchange factor for Ras. The depletion of intracellular pools of guanine nucleotide exchange factors was proposed as the mechanism underlying dominant-negative effects of this mutation with respect to cellular Ras signaling. The failure of G protein subunit dissociation was also observed for the corresponding mutation G226A in Gαs
). The accumulation of the faster-sedimenting complexes resulting from G224A expression suggests intriguing parallels with the model of the persistent association of RasG60A and Gα subunits with their binding partners. Further investigation will be needed to confirm whether analogous molecular mechanisms account for the dominant effects of the switch II glycine mutations. It is remarkable, however, that the impairment of structural flexibility of the switch II region shows a common propensity for dominant-inhibitory effects in distantly related GTPases, indicating a common “weak link” in their molecular structure, despite functional diversification during evolution.
The finding that a subtle alteration in the G domain of mouse Nog1 is sufficient to cause profound consequences for preribosome assembly suggests a potential approach to specifically inhibit ribosome biogenesis in mammalian cells. Given the high evolutionary conservation of Nog1, analogous mutations may also provide a useful tool for studying ribosome assembly in other eukaryotes. It would be interesting to see whether mutations that limit structural transitions in other Obg GTPases would also lead to informative phenotypes. Obg GTPases are found in all living organisms, where these GTPases participate in processes ranging from the assembly and maintenance of ribonucleoprotein complexes to the replication of chromosomes and stress response, offering the possibility of control over a number of important cellular functions.