Farnesol inhibits translation to limit growth and filamentation in C. albicans and S. cerevisiae.

Candida albicans is a polymorphic yeast where the capacity to switch between yeast and filamentous growth is critical for pathogenicity. Farnesol is a quorum-sensing sesquiterpene alcohol that, via regulation of specific signalling and transcription components, inhibits filamentous growth in C. albicans. Here we show that farnesol also inhibits translation at the initiation step in both C. albicans and S. cerevisiae. In contrast to fusel alcohols, that target the eukaryotic initiation factor 2B (eIF2B), farnesol affects the interaction of the mRNA with the small ribosomal subunit leading to reduced levels of the 48S preinitiation ribosomal complex in S. cerevisiae. Therefore, farnesol targets a different step in the translation pathway than fusel alcohols to elicit a completely opposite physiological outcome by negating filamentous growth

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

SRSF1-dependent nuclear export of C9ORF72 repeat-transcripts: targeting toxic gain-of-functions induced by protein sequestration as a selective therapeutic strategy for neuroprotection

Microsatellite repeat expansions cause several incurable and lethal neurodegenerative disorders including ataxias, myotonic dystrophy, Huntington's disease and C9ORF72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Abnormal repeat transcripts generated from the expanded loci are substrates of repeat-associated non-AUG (RAN) translation, an unconventional form of translation leading to the production of polymeric repeat proteins with cytotoxic and aggregating properties. The mechanisms involved in the pathogenesis of microsatellite repeat expansion disorders remain a hotly debated topic. They are shared between toxic loss/gain of functions due to intranuclear RNA foci that sequesters RNA-binding proteins and RAN translation of repeat proteins in the cytoplasm. We recently elucidated the molecular mechanism driving the nuclear export of C9ORF72 repeat transcripts and showed for the first time that this pathway can be manipulated to confer neuroprotection. Strikingly, we discovered that intron-retaining C9ORF72 repeat transcripts hijack the physiological NXF1-dependent export pathway by selective RNA-repeat sequestration of SRSF1. Antagonizing SRSF1 and the nuclear export of C9ORF72 repeat transcripts promoted in turn the survival of patient-derived motor neurons and suppressed neurodegeneration-associated motor deficits in Drosophila (Hautbergue et al. Nature Communications 2017; 8:16063). In this invited Research Highlight review, we aim to place this work in the context of our previous studies on the nuclear export of mRNAs, provide a summary of the published research and highlight the significance of these findings as a novel therapeutic strategy for neuroprotection in C9ORF72-ALS/FTD. In addition, we emphasize that protein sequestration, often thought as of inducing loss-of-function mechanisms, can also trigger unwanted protein interactions and toxic gain-of-functions

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

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

Archetypal transcriptional blocks underpin yeast gene regulation in response to changes in growth conditions

The transcriptional responses of yeast cells to diverse stresses typically include gene activation and repression. Specific stress defense, citric acid cycle and oxidative phosphorylation genes are activated, whereas protein synthesis genes are coordinately repressed. This view was achieved from comparative transcriptomic experiments delineating sets of genes whose expression greatly changed with specific stresses. Less attention has been paid to the biological significance of 1) consistent, albeit modest, changes in RNA levels across multiple conditions, and 2) the global gene expression correlations observed when comparing numerous genome-wide studies. To address this, we performed a meta-analysis of 1379 microarray-based experiments in yeast, and identified 1388 blocks of RNAs whose expression changes correlate across multiple and diverse conditions. Many of these blocks represent sets of functionally-related RNAs that act in a coordinated fashion under normal and stress conditions, and map to global cell defense and growth responses. Subsequently, we used the blocks to analyze novel RNA-seq experiments, demonstrating their utility and confirming the conclusions drawn from the meta-analysis. Our results provide a new framework for understanding the biological significance of changes in gene expression: ‘archetypal’ transcriptional blocks that are regulated in a concerted fashion in response to external stimuli

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

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

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

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