A molecular network of conserved factors keeps ribosomes dormant in the egg

Ribosomes are produced in large quantities during oogenesis and stored in the egg. However, the egg and early embryo are translationally repressed. Using mass-spectrometry and cryo-EM analyses of ribosomes isolated from zebrafish and Xenopus eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules and occupy functionally important sites of the ribosome: a Habp4-eEF2 module that stabilizes ribosome levels and a Dap1b/Dapl1-eIF5a module that represses translation. Dap1b/Dapl1 is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Thus, a developmentally programmed, conserved ribosome state plays a key role in ribosome storage and translational repression in the egg.

Genomic Action of Sigma-1 Receptor Chaperone Relates to Neuropathic Pain

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a “spare nerve injury” (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.

A deep intronic variant activates a pseudoexon in the MTM1 gene in a family with X-linked myotubular myopathy

We report a novel intronic variant in the MTM1 gene in four males in a family with severe X-linked myotubular myopathy. The A>G variant in deep intronic space activates a cryptic 5’ donor splice site resulting in the inclusion of a 48bp pseudoexon into the mature MTM1 mRNA. The variant is present in all affected males, absent in unaffected males and heterozygous in the mother of the affected males. The included intronic sequence contains a premature stop codon and experiments using a translational inhibitor indicate that the mutant mRNAs undergo nonsense-mediate decay. We conclude that affected males produce no, or low, levels of myotubularin-1 protein leading to a severe neonatal myopathy. The study highlights the need to consider non-coding variants in genomic screening in families with X-linked myotubular myopathy.

Bacterial interspecies interactions modulate pH-mediated antibiotic tolerance

Predicting antibiotic efficacy within microbial communities remains highly challenging. Interspecies interactions can impact antibiotic activity through many mechanisms, including alterations to bacterial physiology. Here, we studied synthetic communities constructed from the core members of the fruit fly gut microbiota. Co-culturing of Lactobacillus plantarum with Acetobacter species altered its tolerance to the transcriptional inhibitor rifampin. By measuring key metabolites and environmental pH, we determined that Acetobacter species counter the acidification driven by L. plantarum production of lactate. Shifts in pH were sufficient to modulate L. plantarum tolerance to rifampin and the translational inhibitor erythromycin. A reduction in lag time exiting stationary phase was linked to L. plantarum tolerance to rifampicin, opposite to a previously identified mode of tolerance to ampicillin in E. coli. This mechanistic understanding of the coupling among interspecies interactions, environmental pH, and antibiotic tolerance enables future predictions of growth and the effects of antibiotics in more complex communities.

Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.

A Deep Intronic Variant Activates a Pseudoexon in the MTM1Gene in a Family with X-Linked Myotubular Myopathy

We report a novel intronic variant in the <i>MTM1</i> gene in 4 males in a family with severe X-linked myotubular myopathy. The A&#x3e;G variant in deep intronic space activates a cryptic 5′ donor splice site resulting in the inclusion of a 48-bp pseudoexon into the mature <i>MTM1</i> mRNA. The variant is present in all affected males, absent in unaffected males, and heterozygous in the mother of the affected males. The included intronic sequence contains a premature stop codon, and experiments using a translational inhibitor indicate that the mutant mRNAs undergo nonsense-mediated decay. We conclude that affected males produce no, or low, levels of <i>MTM1</i> mRNA likely leading to a significant reduction of myotubularin-1 protein resulting in the severe neonatal myopathy present in this family. The study highlights the need to consider noncoding variants in genomic screening in families with X-linked myotubular myopathy.

The negative adipogenesis regulator DLK1 is transcriptionally regulated by TIS7 (IFRD1) and translationally by its orthologue SKMc15 (IFRD2)

Delta-like homolog 1 (DLK1), an inhibitor of adipogenesis, controls the cell fate of adipocyte progenitors. Understanding regulatory mechanisms affecting DLK1 levels is therefore of high interest. Here we identify two independent mechanisms, transcriptional and translational, by which TIS7 (IFRD1) and its orthologue SKMc15 (IFRD2) regulate DLK1 levels. Mice deficient in both TIS7 and SKMc15 (dKO) had severely reduced adipose tissue and were resistant to high fat diet-induced obesity. Wnt signaling, a negative regulator of adipocyte differentiation was significantly up regulated in dKO mice. Elevated levels of the Wnt/β-catenin target protein Dlk-1 inhibited the expression of adipogenesis regulators PPARγ and C/EBPα, and fatty acid transporter CD36. Although both, TIS7 and SKMc15, contributed to this phenotype, they utilized two different mechanisms. TIS7 acted by controlling Wnt signaling and thereby transcriptional regulation of Dlk-1. On the other hand, here we provide experimental evidence that SKMc15 acts as a general translational inhibitor affecting DLK-1 protein levels. Our study provides data describing novel mechanisms of DLK1 regulation in adipocyte differentiation involving TIS7 and SKMc15.

4E-BP is a target of the GCN2–ATF4 pathway during Drosophila development and aging

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation–activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

Molecular control of irreversible bistability during trypanosome developmental commitment

The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, proliferation, and transmission of these parasites. One of these, the differentiation of growth-arrested stumpy forms in the mammalian blood into insect-stage procyclic forms, can be induced synchronously in vitro with cis-aconitate. Here, we show that this transition is an irreversible bistable switch, and we map the point of commitment to differentiation after exposure to cis-aconitate. This irreversibility implies that positive feedback mechanisms operate to allow commitment (i.e., the establishment of “memory” of exposure to the differentiation signal). Using the reversible translational inhibitor cycloheximide, we show that this signal memory requires new protein synthesis. We further performed stable isotope labeling by amino acids in cell culture to analyze synchronized parasite populations, establishing the protein and phosphorylation profile of parasites pre- and postcommitment, thereby defining the “commitment proteome.” Functional interrogation of this data set identified Nek-related kinase as the first-discovered protein kinase controlling the initiation of differentiation to procyclic forms.