The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions

The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown.

Compound heterozygous variants in SHQ1 are associated with a spectrum of neurological features, including early-onset dystonia

SHQ1 is essential for biogenesis of H/ACA ribonucleoproteins, a class of molecules important for processing ribosomal RNAs, modifying spliceosomal small nuclear RNAs and stabilizing telomerase. Components of the H/ACA ribonucleoprotein complex have been linked to neurological developmental defects. Here, we report two sibling pairs from unrelated families with compound heterozygous variants in SHQ1. Exome sequencing was used to detect disease causing variants, which were submitted to ‘matching’ platforms linked to MatchMaker Exchange. Phenotype comparisons supported these matches. The affected individuals present with early-onset dystonia, with individuals from one family displaying additional neurological phenotypes, including neurodegeneration. As a result of cerebrospinal fluid studies suggesting possible abnormal dopamine metabolism, a trial of levodopa replacement therapy was started but no clear response was noted. We show that fibroblasts from affected individuals have dramatic loss of SHQ1 protein. Variants from both families were expressed in Saccharomyces cerevisiae, resulting in a strong reduction in H/ACA snoRNA production and remarkable defects in rRNA processing and ribosome formation. Our study identifies SHQ1 as associated with neurological disease, including early-onset dystonia, and begins to delineate the molecular etiology of this novel condition.

Expression and Localization of C-APP and N-APP in Peripheral Neurons of Rats and Crayfish After Axotomy

Nerve injury induces a cascade of molecular-cellular events, leading to neuronal death or survival, where amyloid precursor protein (APP) and its proteolytic products play an important role. We studied the localization and expression of C-APP and N-APP in rat dorsal root ganglia (DRG) with transected sciatic nerve, axotomized crayfish stretch receptor neuron (SRN) and ventral nerve cord (VNC) ganglia with transected connectives. C-APP and N-APP localized predominantly in neurons, not in glial cells. Axotomy increased C-APP and N-APP expression in rat and crayfish neurons. The expression of APP in crustaceans confirms its conservative nature. In DRG, C-APP level was higher in neuronal nuclei than in cytoplasm in 24 hours post-axotomy. N-APP accumulation was not observed in DRG and crayfish neuronal nuclei. SRN axotomy resulted in C-APP and N-APP accumulation in 4–8 hours in perikaryon and its extensions, but only С-APP accumulated in nuclei. This indicates that not the whole APP, but its C-terminal product, AICD, enters the nucleus. Also, there was high level of C-APP in SRN nucleolus, suggesting possible AICD involvement in rRNA synthesis and ribosome formation. The APP accumulation in transected axons confirms its involvement in injury-induced axonal events.

Mosaic changes to the global transcriptome in response to inhibiting ribosome formation versus inhibition of ribosome function

Cell fate is susceptible to several internal and external stresses. Stress resulting from mutations in genes for ribosomal proteins and assembly factors leads to many congenital diseases, collectively called ribosomopathies. Even though such mutations all depress the cell’s protein synthesis capacity, they are manifested in many different phenotypes. This prompted us to use Saccharomyces cerevisiae to explore whether reducing the protein synthesis capacity by different mechanisms result in the same or different changes to the global transcriptome. We have compared the transcriptome after abolishing the assembly of new ribosomes and inhibiting the translocation of ribosomes on the mRNA. Our results show that these alternate obstructions generate different mosaics of expression for several classes of genes, including genes for ribosomal proteins, mitotic cell cycle, cell wall synthesis, and protein transport.

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.

The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

The Nucleolus: A Multiphase Condensate Balancing Ribosome Synthesis and Translational Capacity in Health, Aging and Ribosomopathies

The nucleolus is the largest membrane-less structure in the eukaryotic nucleus. It is involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and is the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the intricate pathophysiological relationship between the nucleolus and protein synthesis has only recently begun to emerge. Here, we provide perspective on new principles governing nucleolar formation and the resulting multiphase organization driven by liquid-liquid phase separation. With recent advances in the structural analysis of ribosome formation, we highlight the current understanding of the step-wise assembly of pre-ribosomal subunits and the quality control required for proper function. Finally, we address how aging affects ribosome genesis and how genetic defects in ribosome formation cause ribosomopathies, complex diseases with a predisposition to cancer.

The GTPase Nog1 couples polypeptide exit tunnel quality control with ribosomal stalk assembly

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the large subunit precursor (pre-60S), these essential steps include eviction of placeholders Arx1 and Mrt4 that prevent premature loading of the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 recruitment to the pre-60S in order to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity extracts the C-terminal tail of Nog1 from the PET, enabling Rei1 to probe PET integrity, and then catalyze Arx1 release. Subsequently, GTPase hydrolysis stimulates Nog1 removal from the pre-60S, permitting Yvh1 to mediate Mrt4 release, and initiate ribosomal stalk assembly. Thus, Nog1 couples quality control and assembly of spatially distant functional centers during ribosome formation.

Magnesium Suppresses Defects in the Formation of 70S Ribosomes as Well as in Sporulation Caused by Lack of Several Individual Ribosomal Proteins

ABSTRACTIndividually, the ribosomal proteins L1, L23, L36, and S6 are not essential for cell proliferation ofBacillus subtilis, but the absence of any one of these ribosomal proteins causes a defect in the formation of the 70S ribosomes and a reduced growth rate. In mutant strains individually lacking these ribosomal proteins, the cellular Mg2+content was significantly reduced. The deletion of YhdP, an exporter of Mg2+, and overexpression of MgtE, the main importer of Mg2+, increased the cellular Mg2+content and restored the formation of 70S ribosomes in these mutants. The increase in the cellular Mg2+content improved the growth rate and the cellular translational activity of the ΔrplA(L1) and the ΔrplW(L23) mutants but did not restore those of the ΔrpmJ(L36) and the ΔrpsF(S6) mutants. The lack of L1 caused a decrease in the production of Spo0A, the master regulator of sporulation, resulting in a decreased sporulation frequency. However, deletion ofyhdPand overexpression ofmgtEincreased the production of Spo0A and partially restored the sporulation frequency in the ΔrplA(L1) mutant. These results indicate that Mg2+can partly complement the function of several ribosomal proteins, probably by stabilizing the conformation of the ribosome.IMPORTANCEWe previously reported that an increase in cellular Mg2+content can suppress defects in 70S ribosome formation and growth rate caused by the absence of ribosomal protein L34. In the present study, we demonstrated that, even in mutants lacking individual ribosomal proteins other than L34 (L1, L23, L36, and S6), an increase in the cellular Mg2+content could restore 70S ribosome formation. Moreover, the defect in sporulation caused by the absence of L1 was also suppressed by an increase in the cellular Mg2+content. These findings indicate that at least part of the function of these ribosomal proteins can be complemented by Mg2+, which is essential for all living cells.