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Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes (1-3). Nuclear export is a key, irreversible step in pre-ribosome maturation (4-8), but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved Saccharomyces cerevisiae GTPase Nug2 (also known as Nog2, and as NGP-1, GNL2 or nucleostemin 2 in human (9)) has a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor (10). Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding before Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre60S remodelling factors, the Real ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre60S particles requires both its [K.sup.+]-dependent GTPase activity and the remodelling ATPase activity of Real. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery. CEN, LEU2, [P.sub. Ref. (21) & This Study GAL1-10]-GFP-realDAA This Study, The conserved GTPase Nug2 (Extended Data Fig. 1) is associated with several pre-60S particles located in the nucleoplasm, but was not detected on particles with a known cytoplasmic location (see [...]

The circularly permuted GTPase YIqF is essential for cell viability and is broadly conserved from Gram-positive bacteria to eukaryotes. We previously reported that YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. Here, we demonstrate that an N-terminal deletion mutant of Y1qF (YlqFΔN10) inhibits cell growth even in the presence of wild-type Y1qF. In contrast to the wild-type protein, the GTPase activity of this mutant was not stimulated by the 50 S subunit and did not dissociate from the premature 50 S subunit. Thus, YlqFΔN10 acts as a competitive inhibitor of wild-type Y1qF. Premature 50 S subunit lacking ribosomal protein L27 and with a reduced amount of L16 accumulated in YlqFΔN10-overexpressing cells and in Y1qF-depleted cells, suggesting that YlqFΔN10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqFΔN10-overexpressing and Y1qF-depleted cells more strongly enhanced the GTPase activity of Y1qF than the mature 50 S subunit of the 70 S ribosome. Collectively, our results indicate that Y1qF is targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA. [ABSTRACT FROM AUTHOR]

Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly. [ABSTRACT FROM AUTHOR]

In yeast, multiprotein bridging factor 1 (Mbf1) has been proposed to function in the integrated stress response (ISR) as a transcriptional coactivator by mediating a direct interaction between general transcription machinery and the process's key effector, Gcn4. However, mounting evidence has demonstrated that Mbf1 (and its human homolog EDF1) is recruited to collided ribosomes, a known activator of the ISR. In this study, we connect these otherwise seemingly disparate functions of Mbf1. Our biochemical and structural analyses reveal that Mbf1 functions as a core ISR factor by interacting with collided ribosomes to mediate Gcn2 activation. We further show that Mbf1 serves no role as a transcriptional coactivator of Gcn4. Instead, Mbf1 is required for optimal stress-induced eukaryotic initiation factor 2α (eIF2α) phosphorylation and downstream de-repression of GCN4 translation. Collectively, our data establish that Mbf1 functions in ISR signaling by acting as a direct sensor of stress-induced ribosome collisions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)