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Ataxin-2 is a conserved translational control protein associated with spinocerebellar ataxia type II (SCA2) and amyotrophic lateral sclerosis (ALS) as well as an important target for ALS therapeutics under development. Despite its clinical and biological significance, Ataxin-2’s activities, mechanisms and functions are not well understood. While Drosophila Ataxin-2 (Atx2) mediates mRNP condensation via a C-terminal intrinsically disordered domain (cIDR), how Ataxin-2 IDRs work with structured (Lsm, Lsm-AD and PAM2) domains to enable positive and negative regulation of target mRNAs remains unclear. Using TRIBE (Targets of RNA-Binding Proteins Identified by Editing) technology, we identified and analysed Atx-2 target mRNAs in the Drosophila brain. We show that Atx2 preferentially interacts with AU-rich elements (AREs) in 3’UTRs and plays a broad role in stabilization of identified target mRNAs. Strikingly, Atx2 interaction with its targets is dependent on the cIDR domain required for neuronal-granule formation. In contrast, Atx2 lacking its Lsm domain not only interacts more efficiently with the target mRNA identified, but also forms larger RNP granules. Providing an extensive dataset of Atx2-interacting brain mRNAs, our results demonstrate that Atx2: (a) interacts with target mRNAs within RNP granules; (b) modulates the turnover of these target mRNAs; (c) has an additional essential role outside of mRNP granules; and (d) contains distinct protein domains that drive or oppose RNP-granule assembly. These findings increase understanding of neuronal translational control mechanisms and inform Ataxin-2-based interventions in development for SCA2 and ALS.

The regulation of stem cell fate is poorly understood. Genetic studies in Caenorhabditis elegans lead to the hypothesis that a conserved cytoplasmic double-negative feedback loop consisting of the RNA-binding protein Trim71 and the let-7 microRNA controls the pluripotency and differentiation of stem cells. Although let-7-microRNA-mediated inhibition of Trim71 promotes differentiation, whether and how Trim71 regulates pluripotency and inhibits the let-7 microRNA are still unknown. Here, we show that Trim71 represses Ago2 mRNA translation in mouse embryonic stem cells. Blocking this repression leads to a specific post-transcriptional increase of mature let-7 microRNAs, resulting in let-7-dependent stemness defects and accelerated differentiation in the stem cells. These results not only support the Trim71-let-7-microRNA bi-stable switch model in controlling stem cell fate, but also reveal that repressing the conserved pro-differentiation let-7 microRNAs at the mature microRNA level by Ago2 availability is critical to maintaining pluripotency.

The regulation of stem cell fate is poorly understood. Genetic studies in Caenorhabditis elegans lead to the hypothesis that a conserved cytoplasmic double-negative feedback loop consisting of the RNA-binding protein Trim71 and the let-7 microRNA controls the pluripotency and differentiation of stem cells. Although let-7-microRNA-mediated inhibition of Trim71 promotes differentiation, whether and how Trim71 regulates pluripotency and inhibits the let-7 microRNA are still unknown. Here, we show that Trim71 represses Ago2 mRNA translation in mouse embryonic stem cells. Blocking this repression leads to a specific post-transcriptional increase of mature let-7 microRNAs, resulting in let-7-dependent stemness defects and accelerated differentiation in the stem cells. These results not only support the Trim71-let-7-microRNA bi-stable switch model in controlling stem cell fate, but also reveal that repressing the conserved pro-differentiation let-7 microRNAs at the mature microRNA level by Ago2 availability is critical to maintaining pluripotency.

Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B. DOI: http://dx.doi.org/10.7554/eLife.24542.001, eLife digest All cells sense and react to changes in the world around them. One way that cells react to threats to their health is by switching off genes required for their normal activity and diverting this energy to switching on genes that deal with the specific stress. A protein called eIF2 controls this general response, which is known as the “integrated stress response”. The eIF2 protein is switched on when it binds to a molecule of GTP and switched off when it binds to a molecule of GDP; these two molecules are swapped by another protein called eIF2B. Previous research revealed that the eIF2B protein attaches to eIF2 that is carrying GDP (also known as eIF2-GDP) and decides whether to switch it to eIF2-GTP or not depending on signals from the integrated stress response. However it was not known if eIF2B could also attach to eIF2-GTP or how this might affect the eIF2 protein, the activity of genes and the integrated stress response. Now, using proteins extracted from baker’s yeast as a model, Jennings et al. studied the interactions between eIF2 and eIF2B. The experiments revealed that, under stressful conditions, eIF2B not only triggers the integrated stress response through eIF2-GDP but also halts any active eIF2-GTP not under the control of this response. Jennings et al. suggest that these two processes represent a fail-safe switch that ensures that the integrated stress responses occurs rapidly whenever the cell is stressed. It is not clear how important this proposed fail-safe switch is for different types of cells, and so further studies will explore this question. In particular, people with mutations in the five genes that encode eIF2B develop a fatal brain disease and have an impaired integrated stress response, and so studies might check to see if some of these mutations affect the fail-safe switch. DOI: http://dx.doi.org/10.7554/eLife.24542.002

Plant immunity is tightly regulated to ensure proper defense against surrounding microbial pathogens without triggering autoimmunity, which negatively impacts plant growth and development. Immune receptor levels are intricately controlled by RNA processing and post-translational modification events, such as ubiquitination. It remains unknown whether, and if yes, how, plant immune receptor homeostasis is regulated at the translational level. From a mutant, snc1-enhancing (muse) forward genetic screen, we identified MUSE11/EXA1, which negatively regulates nucleotide-binding leucine-rich repeat (NLR) receptor mediated defence. EXA1 contains an evolutionarily conserved glycine-tyrosine-phenylalanine (GYF) domain that binds proline-rich sequences. Genetic and biochemical analysis revealed that loss of EXA1 leads to heightened NLR accumulation and enhanced resistance against virulent pathogens. EXA1 also associates with eIF4E initiation factors and the ribosome complex, likely contributing to the proper translation of target proteins. In summary, our study reveals a previously unknown mechanism of regulating NLR homeostasis through translational repression by a GYF protein. DOI: http://dx.doi.org/10.7554/eLife.23684.001

The formation of imprinted memories during a critical period is crucial for vital behaviors, including filial attachment. Yet, little is known about the underlying molecular mechanisms. Using a combination of behavior, pharmacology, in vivo surface sensing of translation (SUnSET) and DiOlistic labeling we found that, translational control by the eukaryotic translation initiation factor 2 alpha (eIF2α) bidirectionally regulates auditory but not visual imprinting and related changes in structural plasticity in chickens. Increasing phosphorylation of eIF2α (p-eIF2α) reduces translation rates and spine plasticity, and selectively impairs auditory imprinting. By contrast, inhibition of an eIF2α kinase or blocking the translational program controlled by p-eIF2α enhances auditory imprinting. Importantly, these manipulations are able to reopen the critical period. Thus, we have identified a translational control mechanism that selectively underlies auditory imprinting. Restoring translational control of eIF2α holds the promise to rejuvenate adult brain plasticity and restore learning and memory in a variety of cognitive disorders. DOI: http://dx.doi.org/10.7554/eLife.17197.001, eLife digest Shortly after hatching, a chick recognizes the sight and sound of its mother and follows her around. This requires a type of learning called imprinting, which only occurs during a short period of time in young life known as the “critical period”. This process has been reported in a variety of birds and other animals where long-term memory formed during a critical period guides vital behaviors. In order to form imprinted memories, neurons must produce new proteins. However, it is not clear how new experiences trigger the production of these proteins during imprinting. Unraveling such mechanisms may help us to develop drugs that can recover plasticity in the adult brain, which could help individuals with brain injuries relearn skills after critical periods are closed. It is possible to imprint newly hatched chicks to arbitrary sounds and visual stimuli by placing the chicks in running wheels and exposing them to repeated noises and videos. Later on, the chicks respond to these stimuli by running towards the screen, mimicking how they would naturally follow their mother. This system allows researchers to measure imprinting in a carefully controlled laboratory setting. A protein called elF2α plays a major role in regulating the production of new proteins and has been shown to be required for the formation of long-term memories in adult rodents. Batista et al. found that elF2α is required to imprint newly hatched chicks to sound. During the critical period, this factor mediates an increase in “memory-spines”, which are small bumps on neurons that are thought to be involved in memory storage. On the other hand, elF2α was not required to imprint newly hatched chicks to visual stimuli, suggesting that there are different pathways involved in regulating imprinting to different senses. Batista et al. also demonstrate that using drugs to increase the activity of eIF2α in older chicks could allow these chicks to be imprinted to new sounds. The next steps following on from this work are to identify proteins that eIF2α regulates to form memories, and to find out why eIF2α is only required to imprint sounds. Future research will investigate the mechanisms that control visual imprinting and how it differs from imprinting to sounds. DOI: http://dx.doi.org/10.7554/eLife.17197.002

Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001, eLife digest To produce a protein, a gene’s DNA is first copied to make molecules of messenger RNA (mRNA). The mRNAs pass through a molecular machine known as the ribosome, which translates the genetic code to make a protein. Not all of an mRNA is translated to make a protein; the “untranslated” regions play crucial roles in regulating how much of the protein is produced. In animals, plants and other eukaryotes, many mRNAs are made up of small pieces that are “spliced” together. During this process, proteins are deposited on the mRNA to mark the splice junctions, which are then cleared when the mRNA is translated. Many different mRNAs can be produced from the same gene by splicing different combinations of RNA pieces. Each of these mRNA “isoforms” can, in principle, contain a unique set of features that control its translation. Hence each mRNA isoform can be translated differently so that different amounts of the corresponding protein product are produced. However, the relationship between the variety of isoforms and the control of translation is complex and not well understood. To address these questions, Floor and Doudna measured the translation of over 60,000 mRNA isoforms made from almost 14,000 human genes. The experiments show that untranslated regions at the end of the mRNA (known as the 3′ end) strongly influence translation, even if the protein coding regions remain the same. Furthermore, the data showed that mRNAs with more splice junctions are translated better, implying an mRNA has some sort of memory of how many junctions it had even after the protein markers have been cleared. Next, Floor and Doudna inserted regulatory sequences from differently translated isoforms into an unrelated “reporter” gene. This dramatically changed the amount of protein produced from the reporter gene, in a manner predicted by the earlier experiments. Untranslated regions at the beginning of the mRNAs (known as the 5′ end) controlled the amount of protein produced from the reporter consistently across different types of cells from the body. On the other hand, the 3′ regions can tune the level of protein production in particular types of cells. Floor and Doudna’s findings demonstrate that differences between mRNA isoforms of a gene can have a big effect on the level of protein production. Changes in the types of mRNA made from a gene are often associated with human diseases, and these findings suggest one reason why. Additionally, the ability to engineer translation of an mRNA using the data is likely to aid the development of mRNA-based therapies. DOI: http://dx.doi.org/10.7554/eLife.10921.002

Growth factor signaling is essential for pattern formation, growth, differentiation, and maintenance of stem cell pluripotency. Nodal-related signaling factors are required for axis formation and germ layer specification from sea urchins to mammals. Maternal transcripts of the zebrafish Nodal factor, Squint (Sqt), are localized to future embryonic dorsal. The mechanisms by which maternal sqt/nodal RNA is localized and regulated have been unclear. Here, we show that maternal control of Nodal signaling via the conserved Y box-binding protein 1 (Ybx1) is essential. We identified Ybx1 via a proteomic screen. Ybx1 recognizes the 3’ untranslated region (UTR) of sqt RNA and prevents premature translation and Sqt/Nodal signaling. Maternal-effect mutations in zebrafish ybx1 lead to deregulated Nodal signaling, gastrulation failure, and embryonic lethality. Implanted Nodal-coated beads phenocopy ybx1 mutant defects. Thus, Ybx1 prevents ectopic Nodal activity, revealing a new paradigm in the regulation of Nodal signaling, which is likely to be conserved. DOI: http://dx.doi.org/10.7554/eLife.00683.001, eLife digest In many organisms, embryonic development is controlled in part by RNAs that are deposited into the egg as it forms inside the mother. These ‘maternal RNAs’ may localize to particular regions of the egg or embryo, where they are then exclusively translated into protein and carry out their specific function. This helps to establish asymmetry in the developing organism—that is, to produce tissues that will eventually become the top or bottom, front or back, and left or right of the organism. One such maternal RNA encodes Nodal, a key signaling molecule that is conserved across vertebrate and some invertebrate organisms. In zebrafish, the equivalent RNA is called squint, and plays an important role in embryonic development. The squint RNA deposited by the mother localizes to the dorsal region—the embryo’s back—and signals that region to make dorsal tissues, but how squint is regulated is not well understood. Now, Kumari et al. identify a protein that controls the positioning of squint RNA, and find that it can also prevent this RNA from being translated into protein. The squint RNA contains a ‘dorsal localization element’ that recruits it to the dorsal cells of the embryo by the 4-cell stage (i.e., within two cell divisions after the egg is fertilized). Kumari et al. identified a protein called Ybx1 that could bind to this element: this protein may help to correctly position RNAs in many other organisms, including fruit flies and mammals. Strikingly, embryos formed abnormally when their maternally derived Ybx1 protein was mutant, and these mutations also prevented the squint RNA from localizing properly. This suggests that maternally derived Ybx1 protein directly regulates the squint RNA. As well as positioning the squint RNA correctly, the embryo must translate this RNA into protein at the right time. In embryos with mutant maternal Ybx1 protein, the Squint protein could be detected at the 16-cell stage, whereas in wild-type embryos this protein is not translated until the 256-cell stage; this indicates that Ybx1 protein might normally repress the translation of the squint RNA. Indeed, Kumari et al. found that Ybx1 binds to another protein—eIF4E—that recruits mRNAs to the ribosome (the cell’s translational machinery). Ybx1 might therefore prevent eIF4E from associating with other components of the ribosomal complex, and initiating the translation of the squint RNA, until additional signals have been received. It will be interesting to determine how widespread this regulatory mechanism is in other organisms. DOI: http://dx.doi.org/10.7554/eLife.00683.002