The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.

Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, Northern Blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with Primer Extension, both approaches often use radioactivity and are time consuming and costly. Here we present "Riboprobing" a reverse transcription-based workflow extended by linker ligation for easy and fast detection and characterization of various pre-rRNA species and their 5` as well as 3` ends. Using standard molecular biology lab equipment, our technique allows reliable discrimination of pre-rRNA species not resolved by Northern Blotting (e.g.: 27SA2, 27SA3 and 27SB). The method can be successfully used for analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA, arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that is lacking most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS for the assembly process.

Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1.

The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases.

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell.

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

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.

Evolution of the analytical scattering model of live Escherichia coli.

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

From Snapshots to Flipbook-Resolving the Dynamics of Ribosome Biogenesis with Chemical Probes.

The synthesis of ribosomes is one of the central and most resource demanding processes in each living cell. As ribosome biogenesis is tightly linked with the regulation of the cell cycle, perturbation of ribosome formation can trigger severe diseases, including cancer. Eukaryotic ribosome biogenesis starts in the nucleolus with pre-rRNA transcription and the initial assembly steps, continues in the nucleoplasm and is finished in the cytoplasm. From start to end, this process is highly dynamic and finished within few minutes. Despite the tremendous progress made during the last decade, the coordination of the individual maturation steps is hard to unravel by a conventional methodology. In recent years small molecular compounds were identified that specifically block either rDNA transcription or distinct steps within the maturation pathway. As these inhibitors diffuse into the cell rapidly and block their target proteins within seconds, they represent excellent tools to investigate ribosome biogenesis. Here we review how the inhibitors affect ribosome biogenesis and discuss how these effects can be interpreted by taking the complex self-regulatory mechanisms of the pathway into account. With this we want to highlight the potential of low molecular weight inhibitors to approach the dynamic nature of the ribosome biogenesis pathway.

Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis.

AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.

Inhibiting eukaryotic ribosome biogenesis: Mining new tools for basic research and medical applications.

The formation of new ribosomes is a fundamental cellular process for each living cell and is tightly interwoven with cell cycle control and proliferation. Minimal disturbances of this pathway can result in ribosomopathies including an increased risk for certain cancer types. Thus, targeting ribosome biogenesis is an emerging strategy in cancer therapy. However, due to its complex nature, we are only at the beginning to understand the dynamics of the ribosome biogenesis pathway. One arising approach that will help us to embrace the tight timely cascade of events that is needed to form a new ribosome is the use of targeted chemical inhibition. However, only very few specific chemical inhibitors of the ribosome biogenesis pathway have been identified so far. Here we review our recently published screen to identify novel inhibitors of the ribosome biogenesis pathway in yeast (Awad et al., 2019, BMC Biology). These inhibitors can provide novel tools for basic research and can serve as starting-points to develop new chemotherapeutics.

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.

Mechanism of completion of peptidyltransferase centre assembly in eukaryotes.

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.

Influence of eukaryotic translation initiation factor 6 on non-small cell lung cancer development and progression.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Dysregulation of protein synthesis plays a major role in carcinogenesis, a process regulated at multiple levels, including translation of mRNA into proteins. Ribosome assembly requires correct association of ribosome subunits, which is ensured by eukaryotic translation initiation factors (eIFs). eIFs have become targets in cancer therapy studies, and promising data on eIF6 in various cancer entities have been reported. Therefore, we hypothesised that eIF6 represents a crossroad for pulmonary carcinogenesis. High levels of eIF6 are associated with shorter patient overall survival in adenocarcinoma (ADC), but not in squamous cell carcinoma (SQC) of the lung. We demonstrate significantly higher protein expression of eIF6 in ADC and SQC than in healthy lung tissue based on immunohistochemical data from tissue microarrays (TMAs) and on fresh frozen lung tissue. Depletion of eIF6 in ADC and SQC lung cancer cell lines inhibited cell proliferation and induced apoptosis. Knockdown of eIF6 led to pre-rRNA processing and ribosomal 60S maturation defects. Our data indicate that eIF6 is upregulated in NSCLC, suggesting an important contribution of eIF6 to the development and progression of NSCLC and a potential for new treatment strategies against NSCLC.

Viewing pre-60S maturation at a minute's timescale.

The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation.

Separation of low and high grade colon and rectum carcinoma by eukaryotic translation initiation factors 1, 5 and 6.

Colorectal cancer (CRC) is the third most common cause of cancer related death worldwide. Furthermore, with more than 1.2 million cases registered per year, it constitutes the third most frequent diagnosed cancer entity worldwide. Deregulation of protein synthesis has received considerable attention as a major step in cancer development and progression. Eukaryotic translation initiation factors (eIFs) are involved in the regulation of protein synthesis and are functionally linked to the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. The identification of factors accounting for colorectal carcinoma (CRC) development is a major gap in the field. Besides the importance of eIF3 subunits and the eIF4 complex, eIF1, eIF5 and eIF6 were found to be altered in primary and metastatic CRC. We observed significant difference in the expression profile between low and high grade CRC. eIF1, eIF5 and eIF6 are involved in translational control in CRC. Our findings also indicate a probable clinical impact when separating them into low and high grade colon and rectum carcinoma. eIF and mTOR expression were analysed on protein and mRNA level in primary low and high grade colon carcinoma (CC) and rectum carcinoma (RC) samples in comparison to non-neoplastic tissue without any disease-related pathology. To assess the therapeutic potential of targeting eIF1, eIF5 and eIF6 siRNA knockdown in HCT116 and HT29 cells was performed. We evaluated the eIF knockdown efficacy on protein and mRNA level and investigated proliferation, apoptosis, invasion, as well as colony forming and polysome associated fractions. These results indicate that eIFs, in particular eIF1, eIF5 and eIF6 play a major role in translational control in colon and rectum cancer.

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.

Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1.

After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/ß pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein.

Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity.

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.

Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation.

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.

Notch4 Signaling Induces a Mesenchymal-Epithelial-like Transition in Melanoma Cells to Suppress Malignant Behaviors.

The effects of Notch signaling are context-dependent and both oncogenic and tumor-suppressive functions have been described. Notch signaling in melanoma is considered oncogenic, but clinical trials testing Notch inhibition in this malignancy have not proved successful. Here, we report that expression of the constitutively active intracellular domain of Notch4 (N4ICD) in melanoma cells triggered a switch from a mesenchymal-like parental phenotype to an epithelial-like phenotype. The epithelial-like morphology was accompanied by strongly reduced invasive, migratory, and proliferative properties concomitant with the downregulation of epithelial-mesenchymal transition markers Snail2 (SNAI2), Twist1, vimentin (VIM), and MMP2 and the reexpression of E-cadherin (CDH1). The N4ICD-induced phenotypic switch also resulted in significantly reduced tumor growth in vivo Immunohistochemical analysis of primary human melanomas and cutaneous metastases revealed a significant correlation between Notch4 and E-cadherin expression. Mechanistically, we demonstrate that N4ICD induced the expression of the transcription factors Hey1 and Hey2, which bound directly to the promoter regions of Snail2 and Twist1 and repressed gene transcription, as determined by EMSA and luciferase assays. Taken together, our findings indicate a role for Notch4 as a tumor suppressor in melanoma, uncovering a potential explanation for the poor clinical efficacy of Notch inhibitors observed in this setting. Cancer Res; 76(7); 1690-7. ©2016 AACR.

eIF3a is over-expressed in urinary bladder cancer and influences its phenotype independent of translation initiation.

PURPOSE: The eukaryotic translation initiation factor (eIF) 3a, the largest subunit of the eIF3 complex, is a key functional entity in ribosome establishment and translation initiation. In the past, aberrant eIF3a expression has been linked to the pathology of various cancer types but, so far, its expression has not been investigated in transitional cell carcinomas. Here, we investigated the impact of eIF3 expression on urinary bladder cancer (UBC) cell characteristics and UBC patient survival. METHODS AND RESULTS: eIF3a expression was reduced through inducible knockdown in the UBC-derived cell lines RT112, T24, 5637 and HT1197. As a consequence of eIF3a down-regulation, UBC cell proliferation, clonogenic potential and motility were found to be decreased and, concordantly, UBC tumour cell growth rates were found to be impaired in xenotransplanted mice. Polysomal profiling revealed that reduced eIF3a levels increased the abundance of 80S ribosomes, rather than impairing translation initiation. Microarray-based gene expression and ontology analyses revealed broad effects of eIF3a knockdown on the transcriptome. Analysis of eIF3a expression in primary formalin-fixed paraffin embedded UBC samples of 198 patients revealed that eIF3a up-regulation corresponds to tumour grade and that high eIF3a expression corresponds to longer overall survival rates of patients with low grade tumours. CONCLUSIONS: From our results we conclude that eIF3a expression may have a profound effect on the UBC phenotype and, in addition, may serve as a prognostic marker for low grade UBCs.