Translation factor and RNA binding protein mRNA interactomes support broader RNA regulons for posttranscriptional control.

The regulation of translation provides a rapid and direct mechanism to modulate the cellular proteome. In eukaryotes, an established model for the recruitment of ribosomes to mRNA depends upon a set of conserved translation initiation factors. Nevertheless, how cells orchestrate and define the selection of individual mRNAs for translation, as opposed to other potential cytosolic fates, is poorly understood. We have previously found significant variation in the interaction between individual mRNAs and an array of translation initiation factors. Indeed, mRNAs can be separated into different classes based upon these interactions to provide a framework for understanding different modes of translation initiation. Here, we extend this approach to include new mRNA interaction profiles for additional proteins involved in shaping the cytoplasmic fate of mRNAs. This work defines a set of seven mRNA clusters, based on their interaction profiles with 12 factors involved in translation and/or RNA binding. The mRNA clusters share both physical and functional characteristics to provide a rationale for the interaction profiles. Moreover, a comparison with mRNA interaction profiles from a host of RNA binding proteins suggests that there are defined patterns in the interactions of functionally related mRNAs. Therefore, this work defines global cytoplasmic mRNA binding modules that likely coordinate the synthesis of functionally related proteins.

Mechanisms of repeat-associated non-AUG translation in neurological microsatellite expansion disorders.

Repeat-associated non-AUG (RAN) translation was discovered in 2011 in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). This non-canonical form of translation occurs in all reading frames from both coding and non-coding regions of sense and antisense transcripts carrying expansions of trinucleotide to hexanucleotide repeat sequences. RAN translation has since been reported in 7 of the 53 known microsatellite expansion disorders which mainly present with neurodegenerative features. RAN translation leads to the biosynthesis of low-complexity polymeric repeat proteins with aggregating and cytotoxic properties. However, the molecular mechanisms and protein factors involved in assembling functional ribosomes in absence of canonical AUG start codons remain poorly characterised while secondary repeat RNA structures play key roles in initiating RAN translation. Here, we briefly review the repeat expansion disorders, their complex pathogenesis and the mechanisms of physiological translation initiation together with the known factors involved in RAN translation. Finally, we discuss research challenges surrounding the understanding of pathogenesis and future directions that may provide opportunities for the development of novel therapeutic approaches for this group of incurable neurodegenerative diseases.

Astrocyte adenosine deaminase loss increases motor neuron toxicity in amyotrophic lateral sclerosis.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

Proteomic and cellular localisation studies suggest non-tight junction cytoplasmic and nuclear roles for occludin in astrocytes.

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states.

The yeast La related protein Slf1p is a key activator of translation during the oxidative stress response.

The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p 'closed loop' complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control.

The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3' UTR motif. Caf20p binds all tested motif-containing 3' UTRs. Caf20p and the 3'UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3'UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.

Puf3p induces translational repression of genes linked to oxidative stress.

In response to stress, the translation of many mRNAs in yeast can change in a fashion discordant with the general repression of translation. Here, we use machine learning to mine the properties of these mRNAs to determine specific translation control signals. We find a strong association between transcripts acutely translationally repressed under oxidative stress and those associated with the RNA-binding protein Puf3p, a known regulator of cellular mRNAs encoding proteins targeted to mitochondria. Under oxidative stress, a PUF3 deleted strain exhibits more robust growth than wild-type cells and the shift in translation from polysomes to monosomes is attenuated, suggesting puf3Δ cells perceive less stress. In agreement, the ratio of reduced:oxidized glutathione, a major antioxidant and indicator of cellular redox state, is increased in unstressed puf3Δ cells but remains lower under stress. In untreated conditions, Puf3p migrates with polysomes rather than ribosome-free fractions, but this is lost under stress. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) of Puf3p targets following affinity purification shows Puf3p-mRNA associations are maintained or increased under oxidative stress. Collectively, these results point to Puf3p acting as a translational repressor in a manner exceeding the global translational response, possibly by temporarily limiting synthesis of new mitochondrial proteins as cells adapt to the stress.

PKA isoforms coordinate mRNA fate during nutrient starvation.

A variety of stress conditions induce mRNA and protein aggregation into mRNA silencing foci, but the signalling pathways mediating these responses are still elusive. Previously we demonstrated that PKA catalytic isoforms Tpk2 and Tpk3 localise with processing and stress bodies in Saccharomyces cerevisiae. Here, we show that Tpk2 and Tpk3 are associated with translation initiation factors Pab1 and Rps3 in exponentially growing cells. Glucose starvation promotes the loss of interaction between Tpk and initiation factors followed by their accumulation into processing bodies. Analysis of mutants of the individual PKA isoform genes has revealed that the TPK3 or TPK2 deletion affects the capacity of the cells to form granules and arrest translation properly in response to glucose starvation or stationary phase. Moreover, we demonstrate that PKA controls Rpg1 and eIF4G(1) protein abundance, possibly controlling cap-dependent translation. Taken together, our data suggest that the PKA pathway coordinates multiple stages in the fate of mRNAs in association with nutritional environment and growth status of the cell.

From use of omics to systems biology: Identifying therapeutic targets for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous progressive neurodegenerative disorder with available treatments such as riluzole and edaravone extending survival by an average of 3-6 months. The lack of highly effective, widely available therapies reflects the complexity of ALS. Omics technologies, including genomics, transcriptomic and proteomics have contributed to the identification of biological pathways dysregulated and targeted by therapeutic strategies in preclinical and clinical trials. Integrating clinical, environmental and neuroimaging information with omics data and applying a systems biology approach can further improve our understanding of the disease with the potential to stratify patients and provide more personalised medicine. This chapter will review the omics technologies that contribute to a systems biology approach and how these components have assisted in identifying therapeutic targets. Current strategies, including the use of genetic screening and biosampling in clinical trials, as well as the future application of additional technological advances, will also be discussed.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.

Atypical TDP-43 protein expression in an ALS pedigree carrying a p.Y374X truncation mutation in TARDBP.

We describe an autosomal dominant, multi-generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co-segregates with a heterozygous p.Y374X nonsense mutation within TDP-43. Mislocalization of TDP-43 and formation of insoluble TDP-43-positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP-43 pathology within lower motor neurons, but classical TDP-43-positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP-43 protein expression is reduced compared to wild-type controls. Despite absence of TDP-43-positive inclusions we confirmed deficient TDP-43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP-43 protein species but not typical C-terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP-43 combined with atypical TDP-43 protein species and absent C-terminal fragments extends the molecular phenotypes associated with TDP-43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP-43-mediated toxicity.

The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function.

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.

A Y374X TDP43 truncation leads to an altered metabolic profile in amyotrophic lateral sclerosis fibroblasts driven by pyruvate and TCA cycle intermediate alterations.

A p.Y374X truncation in TARDBP was recently shown to reduce expression of TDP43 in fibroblasts isolated from ALS cases. In this follow up study focused on assessing the downstream phenotypic consequences of loss of TDP43 in the context of the truncation, we have shown a striking effect on the fibroblast metabolic profile. Phenotypic metabolic screening uncovered a distinct metabolic profile in TDP43-Y374X fibroblasts compared to controls, which was driven by alterations in key metabolic checkpoint intermediates including pyruvate, alpha-ketoglutarate and succinate. These metabolic alterations were confirmed using transcriptomics and bioenergetic flux analysis. These data suggest that TDP43 truncation directly compromises glycolytic and mitochondrial function, identifying potential therapeutic targets for mitigating the effects of TDP43-Y374X truncation.

A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins.

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies.

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins.

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

RNA Helicases in Microsatellite Repeat Expansion Disorders and Neurodegeneration.

Short repeated sequences of 3-6 nucleotides are causing a growing number of over 50 microsatellite expansion disorders, which mainly present with neurodegenerative features. Although considered rare diseases in relation to the relatively low number of cases, these primarily adult-onset conditions, often debilitating and fatal in absence of a cure, collectively pose a large burden on healthcare systems in an ageing world population. The pathological mechanisms driving disease onset are complex implicating several non-exclusive mechanisms of neuronal injury linked to RNA and protein toxic gain- and loss- of functions. Adding to the complexity of pathogenesis, microsatellite repeat expansions are polymorphic and found in coding as well as in non-coding regions of genes. They form secondary and tertiary structures involving G-quadruplexes and atypical helices in repeated GC-rich sequences. Unwinding of these structures by RNA helicases plays multiple roles in the expression of genes including repeat-associated non-AUG (RAN) translation of polymeric-repeat proteins with aggregating and cytotoxic properties. Here, we will briefly review the pathogenic mechanisms mediated by microsatellite repeat expansions prior to focus on the RNA helicases eIF4A, DDX3X and DHX36 which act as modifiers of RAN translation in C9ORF72-linked amyotrophic lateral sclerosis/frontotemporal dementia (C9ORF72-ALS/FTD) and Fragile X-associated tremor/ataxia syndrome (FXTAS). We will further review the RNA helicases DDX5/17, DHX9, Dicer and UPF1 which play additional roles in the dysregulation of RNA metabolism in repeat expansion disorders. In addition, we will contrast these with the roles of other RNA helicases such as DDX19/20, senataxin and others which have been associated with neurodegeneration independently of microsatellite repeat expansions. Finally, we will discuss the challenges and potential opportunities that are associated with the targeting of RNA helicases for the development of future therapeutic approaches.

Loss of TMEM106B exacerbates C9ALS/FTD DPR pathology by disrupting autophagosome maturation.

Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.

Overlapping regions of Caf20 mediate its interactions with the mRNA-5'cap-binding protein eIF4E and with ribosomes.

By interacting with the mRNA 5' cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.

Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease.

Astrocytes are highly specialised cells, responsible for CNS homeostasis and neuronal activity. Lack of human in vitro systems able to recapitulate the functional changes affecting astrocytes during ageing represents a major limitation to studying mechanisms and potential therapies aiming to preserve neuronal health. Here, we show that induced astrocytes from fibroblasts donors in their childhood or adulthood display age-related transcriptional differences and functionally diverge in a spectrum of age-associated features, such as altered nuclear compartmentalisation, nucleocytoplasmic shuttling properties, oxidative stress response and DNA damage response. Remarkably, we also show an age-related differential response of induced neural progenitor cells derived astrocytes (iNPC-As) in their ability to support neurons in co-culture upon pro-inflammatory stimuli. These results show that iNPC-As are a renewable, readily available resource of human glia that retain the age-related features of the donor fibroblasts, making them a unique and valuable model to interrogate human astrocyte function over time in human CNS health and disease.