The C-terminal tail of ribosomal protein Rps15 is engaged in cytoplasmic pre-40S maturation.

The small ribosomal subunit protein Rps15/uS19 is involved in early nucleolar ribosome biogenesis and subsequent nuclear export of pre-40S particles to the cytoplasm. In addition, the C-terminal tail of Rps15 was suggested to play a role in mature ribosomes, namely during translation elongation. Here, we show that Rps15 not only functions in nucleolar ribosome assembly but also in cytoplasmic pre-40S maturation, which is indicated by a strong genetic interaction between Rps15 and the 40S assembly factor Ltv1. Specifically, mutations either in the globular or C-terminal domain of Rps15 when combined with the non-essential ltv1 null allele are lethal or display a strong growth defect. However, not only rps15 ltv1 double mutants but also single rps15 C-terminal deletion mutants exhibit an accumulation of the 20S pre-rRNA in the cytoplasm, indicative of a cytoplasmic pre-40S maturation defect. Since in pre-40S particles, the C-terminal tail of Rps15 is positioned between assembly factors Rio2 and Tsr1, we further tested whether Tsr1 is genetically linked to Rps15, which indeed could be demonstrated. Thus, the integrity of the Rps15 C-terminal tail plays an important role during late pre-40S maturation, perhaps in a quality control step to ensure that only 40S ribosomal subunits with functional Rps15 C-terminal tail can efficiently enter translation. As mutations in the C-terminal tail of human RPS15 have been observed in connection with chronic lymphocytic leukaemia, it is possible that apart from defects in translation, an impaired late pre-40S maturation step in the cytoplasm could also be a reason for this disease.

Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine.

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

Inhibiting eukaryotic ribosome biogenesis.

BACKGROUND: Ribosome biogenesis is a central process in every growing cell. In eukaryotes, it requires more than 250 non-ribosomal assembly factors, most of which are essential. Despite this large repertoire of potential targets, only very few chemical inhibitors of ribosome biogenesis are known so far. Such inhibitors are valuable tools to study this highly dynamic process and elucidate mechanistic details of individual maturation steps. Moreover, ribosome biogenesis is of particular importance for fast proliferating cells, suggesting its inhibition could be a valid strategy for treatment of tumors or infections. RESULTS: We systematically screened ~ 1000 substances for inhibitory effects on ribosome biogenesis using a microscopy-based screen scoring ribosomal subunit export defects. We identified 128 compounds inhibiting maturation of either the small or the large ribosomal subunit or both. Northern blot analysis demonstrates that these inhibitors cause a broad spectrum of different rRNA processing defects. CONCLUSIONS: Our findings show that the individual inhibitors affect a wide range of different maturation steps within the ribosome biogenesis pathway. Our results provide for the first time a comprehensive set of inhibitors to study ribosome biogenesis by chemical inhibition of individual maturation steps and establish the process as promising druggable pathway for chemical intervention.

Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME.

Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.