FOXO-mediated repression of Dicer1 regulates metabolism, stress resistance, and longevity in Drosophila.

The adipose tissue plays a crucial role in metabolism and physiology, affecting animal lifespan and susceptibility to disease. In this study, we present evidence that adipose Dicer1 (Dcr-1), a conserved type III endoribonuclease involved in miRNA processing, plays a crucial role in the regulation of metabolism, stress resistance, and longevity. Our results indicate that the expression of Dcr-1 in murine 3T3L1 adipocytes is responsive to changes in nutrient levels and is subject to tight regulation in the Drosophila fat body, analogous to human adipose and hepatic tissues, under various stress and physiological conditions such as starvation, oxidative stress, and aging. The specific depletion of Dcr-1 in the Drosophila fat body leads to changes in lipid metabolism, enhanced resistance to oxidative and nutritional stress, and is associated with a significant increase in lifespan. Moreover, we provide mechanistic evidence showing that the JNK-activated transcription factor FOXO binds to conserved DNA-binding sites in the dcr-1 promoter, directly repressing its expression in response to nutrient deprivation. Our findings emphasize the importance of FOXO in controlling nutrient responses in the fat body by suppressing Dcr-1 expression. This mechanism coupling nutrient status with miRNA biogenesis represents a novel and previously unappreciated function of the JNK-FOXO axis in physiological responses at the organismal level.

Smaug1 membrane-less organelles respond to AMPK and mTOR and affect mitochondrial function.

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused.

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Life and Work of Stress Granules and Processing Bodies: New Insights into Their Formation and Function.

The dynamic formation of stress granules (SGs), processing bodies (PBs), and related RNA organelles regulates diverse cellular processes, including the coordination of functionally connected messengers, the translational regulation at the synapse, and the control of viruses and retrotransposons. Recent studies have shown that pyruvate kinase and other enzymes localize in SGs and PBs, where they become protected from stress insults. These observations may have implications for enzyme regulation and metabolic control exerted by RNA-based organelles. The formation of these cellular bodies is governed by liquid-liquid phase separation (LLPS) processes, and it needs to be strictly controlled to prevent pathogenic aggregation. The intracellular concentration of key metabolites, such as ATP and sterol derivatives, may influence protein solubility, thus affecting the dynamics of liquid organelles. LLPS in vitro depends on the thermal diffusion of macromolecules, which is limited inside cells, where the condensation and dissolution of membrane-less organelles are helped by energy-driven processes. The active transport by the retrograde motor dynein helps SG assembly, whereas the anterograde motor kinesin mediates SG dissolution; a tug of war between these two molecular motors allows transient SG formation. There is evidence that the efficiency of dynein-mediated transport increases with the number of motor molecules associated with the cargo. The dynein-dependent transport may be influenced by cargo size as larger cargos can load a larger number of motors. We propose a model based on this emergent property of dynein motors, which would be collectively stronger during SG condensation and weaker during SG breakdown, thus allowing kinesin-mediated dispersion.

Two Arabidopsis late pollen transcripts are detected in cytoplasmic granules.

Many of mRNAs synthesized during pollen development are translated after germination, and we hypothesize that they are stored in cytoplasmic granules. We analyzed the cellular localization of the SKS14 and AT59 Arabidopsis mRNAs, which are orthologues of the tobacco NTP303 and tomato LAT59 pollen mRNAs, respectively, by artificially labeling the transcripts with a MS2-GFP chimera. A MATLAB-automated image analysis helped to identify the presence of cytoplasmic SKS14 and AT59 mRNA granules in mature pollen grains. These mRNA granules partially colocalized with VCS and DCP1, two processing body (PB) proteins. Finally, we found a temporal correlation between SKS14 protein accumulation and the disappearance of SKS14 mRNA granules during pollen germination. These results contribute to unveil a mechanism for translational regulation in Arabidopsis thaliana pollen.

Synaptic control of mRNA translation by reversible assembly of XRN1 bodies.

Repression of mRNA translation is linked to the formation of specific cytosolic foci such as stress granules and processing bodies, which store or degrade mRNAs. In neurons, synaptic activity regulates translation at the post-synapse and this is important for plasticity. N-methyl-D-aspartate (NMDA) receptor stimulation downregulates translation, and we speculate that this is linked to the formation of unknown mRNA-silencing foci. Here, we show that the 5'-3' exoribonuclease XRN1 forms discrete clusters associated with the post-synapse that are different from processing bodies or stress granules, and we named them synaptic XRN1 bodies (SX-bodies). Using primary neurons, we found that the SX-bodies respond to synapse stimulation and that their formation correlates inversely with the local translation rate. SX-bodies increase in size and number upon NMDA stimulation, and metabotropic glutamate receptor activation provokes SX-body dissolution, along with increased translation. The response is specific and the previously described Smaug1 foci and FMRP granules show a different response. Finally, XRN1 knockdown impairs the translational repression triggered by NMDA. Collectively, these observations support a role for the SX-bodies in the reversible masking and silencing of mRNAs at the synapse.

Translation and silencing in RNA granules: a tale of sand grains.

The transcriptome at the synapse consists of thousands of messengers encoding several cellular functions, including a significant number of receptors and ion channels and associated proteins. The concerted translational regulation of all these molecules contributes to the dynamic control of synaptic strength. Cumulative evidence supports that dendritic RNA granules and mRNA-silencing foci play an important role in translational regulation. Several relevant RBPs - FMRP; FUS/TLS; TDP-43; Staufen; Smaug; Pumilio; CPEB; HuD; ZBP1; and DDX6 among others - form granules that contain dormant mRNAs repressed by multiple pathways. Recent reports indicate that dendritic granules may contain stalled polysomes, and furthermore, active translation may occur in association with RNA granules. Here, we discuss the molecules and pathways involved in this continuum of RNA granules that contain masked mRNAs, mRNAs trapped in inactive polysomes or mRNAs engaged in translation.

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth.

Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase.

Synaptic activity regulated mRNA-silencing foci for the fine tuning of local protein synthesis at the synapse.

The regulated synthesis of specific proteins at the synapse is important for neuron plasticity, and several localized mRNAs are translated upon specific stimulus. Repression of mRNA translation is linked to the formation of mRNA-silencing foci, including Processing Bodies (PBs) and Stress Granules (SGs), which are macromolecular aggregates that harbor silenced messengers and associated proteins. In a recent work, we identified a kind of mRNA-silencing foci unique to neurons, termed S-foci, that contain the post-transcriptional regulator Smaug1/SAMD4. Upon specific synaptic stimulation, the S-foci dissolve and release mRNAs to allow their translation, paralleling the cycling of mRNAs between PBs and polysomes in other cellular contexts. Smaug 1 and other proteins involved in mRNA regulation in neurons contain aggregation domains distinct from their RNA binding motifs, and we speculate that self-aggregation helps silencing and transport. In addition to S-foci and PBs, other foci formed by distinct RNA binding proteins, such as TDP-43 and FMRP among others, respond dynamically to specific synaptic stimuli. We propose the collective name of synaptic activity-regulated mRNA silencing (SyAS) foci for these RNP aggregates that selectively respond to distinct stimulation patterns and contribute to the fine-tuning of local protein synthesis at the synapse.

Junin virus infection impairs stress-granule formation in Vero cells treated with arsenite via inhibition of eIF2α phosphorylation.

Stress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Junín (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly. We found that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells.

Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

Dynein and kinesin regulate stress-granule and P-body dynamics.

Stress granules (SGs) and P-bodies (PBs) are related cytoplasmic structures harboring silenced mRNAs. SGs assemble transiently upon cellular stress, whereas PBs are constitutive and are further induced by stress. Both foci are highly dynamic, with messenger ribonucleoproteins (mRNPs) and proteins rapidly shuttling in and out. Here, we show that impairment of retrograde transport by knockdown of mammalian dynein heavy chain 1 (DHC1) or bicaudal D1 (BicD1) inhibits SG formation and PB growth upon stress, without affecting protein-synthesis blockage. Conversely, impairment of anterograde transport by knockdown of kinesin-1 heavy chain (KIF5B) or kinesin light chain 1 (KLC1) delayed SG dissolution. Strikingly, SG dissolution is not required to restore translation. Simultaneous knockdown of dynein and kinesin reverted the effect of single knockdowns on both SGs and PBs, suggesting that a balance between opposing movements driven by these molecular motors governs foci formation and dissolution. Finally, we found that regulation of SG dynamics by dynein and kinesin is conserved in Drosophila.

Mammalian Staufen 1 is recruited to stress granules and impairs their assembly.

Stress granules are cytoplasmic mRNA-silencing foci that form transiently during the stress response. Stress granules harbor abortive translation initiation complexes and are in dynamic equilibrium with translating polysomes. Mammalian Staufen 1 (Stau1) is a ubiquitous double-stranded RNA-binding protein associated with polysomes. Here, we show that Stau1 is recruited to stress granules upon induction of endoplasmic reticulum or oxidative stress as well in stress granules induced by translation initiation blockers. We found that stress granules lacking Stau1 formed in cells depleted of this molecule, indicating that Stau1 is not an essential component of stress granules. Moreover, Stau1 knockdown facilitated stress granule formation upon stress induction. Conversely, transient transfection of Stau1 impaired stress granule formation upon stress or pharmacological initiation arrest. The inhibitory capacity of Stau1 mapped to the amino-terminal half of the molecule, a region known to bind to polysomes. We found that the fraction of polysomes remaining upon stress induction was enriched in Stau1, and that Stau1 overexpression stabilized polysomes against stress. We propose that Stau1 is involved in recovery from stress by stabilizing polysomes, thus helping stress granule dissolution.

Mammalian Smaug is a translational repressor that forms cytoplasmic foci similar to stress granules.

Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile alpha motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons.

Staufen recruitment into stress granules does not affect early mRNA transport in oligodendrocytes.

Staufen is a conserved double-stranded RNA-binding protein required for mRNA localization in Drosophila oocytes and embryos. The mammalian homologues Staufen 1 and Staufen 2 have been implicated in dendritic RNA targeting in neurons. Here we show that in rodent oligodendrocytes, these two proteins are present in two independent sets of RNA granules located at the distal myelinating processes. A third kind of RNA granules lacks Staufen and contains major myelin mRNAs. Myelin Staufen granules associate with microfilaments and microtubules, and their subcellular distribution is affected by polysome-disrupting drugs. Under oxidative stress, both Staufen 1 and Staufen 2 are recruited into stress granules (SGs), which are stress-induced organelles containing transiently silenced messengers. Staufen SGs contain the poly(A)-binding protein (PABP), the RNA-binding proteins HuR and TIAR, and small but not large ribosomal subunits. Staufen recruitment into perinuclear SGs is paralleled by a similar change in the overall localization of polyadenylated RNA. Under the same conditions, the distribution of recently transcribed and exported mRNAs is not affected. Our results indicate that Staufen 1 and Staufen 2 are novel and ubiquitous SG components and suggest that Staufen RNPs are involved in repositioning of most polysomal mRNAs, but not of recently synthesized transcripts, during the stress response.