Detecting and quantifying stress granules in tissues of multicellular organisms with the Obj.MPP analysis tool

International audienceStress Granules (SGs) are macromolecular assemblies induced by stress and composed of proteins and mRNAs stalled in translation initiation. SGs play an important role in the response to stress and in the modulation of signaling pathways. Furthermore, these structures are related to the pathological ribonucleoprotein (RNP) aggregates found in neurodegenerative disease contexts, highlighting the need to understand how they are formed and recycled in normal and pathological contexts. Although genetically tractable multicellular organisms have been key in identifying modifiers of RNP aggregate toxicity, in vivo analysis of SG properties and regulation has lagged behind, largely due to the difficulty of detecting SG from images of intact tissues. Here, we describe the object detector software Obj.MPP and show how it overcomes the limits of classical object analyzers to extract the properties of SGs from wide-field and confocal images of respectively C. elegans and Drosophila tissues. We demonstrate that Obj.MPP enables the identification of genes modulating the assembly of endogenous and pathological SGs, and thus that it will be useful in the context of future genetic screens and in vivo studies. This article is protected by copyright. All rights reserved

C. elegans ATAD-3 Is Essential for Mitochondrial Activity and Development

Contains fulltext : 80701.pdf (publisher's version ) (Open Access)BACKGROUND: Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved. RESULTS: Here, we characterize the Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity. CONCLUSIONS: In summary, our data clearly indicate that ATAD-3 is essential for C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages

Stress granules regulate stress-induced paraspeckle assembly

Eukaryotic cells contain a variety of RNA-protein macrocomplexes termed RNP granules. Different types of granules share multiple protein components; however, the crosstalk between spatially separated granules remains unaddressed. Paraspeckles and stress granules (SGs) are prototypical RNP granules localized exclusively in the nucleus and cytoplasm, respectively. Both granules are implicated in human diseases, such as amyotrophic lateral sclerosis. We characterized the composition of affinity-purified paraspeckle-like structures and found a significant overlap between the proteomes of paraspeckles and SGs. We further show that paraspeckle hyperassembly is typical for cells subjected to SG-inducing stresses. Using chemical and genetic disruption of SGs, we demonstrate that formation of microscopically visible SGs is required to trigger and maintain stress-induced paraspeckle assembly. Mechanistically, SGs may sequester negative regulators of paraspeckle formation, such as UBAP2L, alleviating their inhibitory effect on paraspeckles. Our study reveals a novel function for SGs as positive regulators of nuclear RNP granule assembly and suggests a role for disturbed SG-paraspeckle crosstalk in human disease

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium

The multiscale and multiphase organization of the transcriptome

Gene expression must be co-ordinated to cellular activity. From transcription to decay, the expression of millions of RNA molecules is highly synchronized. RNAs are covered by proteins that regulate every aspect of their cellular life: expression, storage, translational status, localization, and decay. Many RNAs and their associated regulatory proteins can coassemble to condense into liquid droplets, viscoelastic hydrogels, freeze into disorganized glass-like aggregates, or harden into quasi-crystalline solids. Phase separations provide a framework for transcriptome organization where the single functional unit is no longer a transcript but instead an RNA regulon. Here, we will analyze the interaction networks that underlie RNA super-assemblies, assess the complex multiscale, multiphase architecture of the transcriptome, and explore how the biophysical state of an RNA molecule can define its fate. Phase separations are emerging as critical routes for the epitranscriptomic control of gene expression.</jats:p

The multiscale and multiphase organization of the transcriptome

International audienceGene expression must be coordinated to cellular activity. From transcription to decay, the expression of millions of RNA molecules is highly synchronized. RNAs are covered by proteins that regulate every aspect of their cellular life: expression, storage, translational status, localization, and decay. Many RNAs and their associated regulatory proteins can coassemble to condense into liquid droplets, viscoelastic hydrogels, freeze into disorganized glass-like aggregates, or harden into quasi-crystalline solids. Phase separations provide a framework for transcriptome organization where the single functional unit is no longer a transcript but instead an RNA regulon. Here, we will analyze the interaction networks that underlie RNA super-assemblies, assess the complex multiscale, multiphase architecture of the transcriptome, and explore how the biophysical state of an RNA molecule can define its fate. Phase separations are emerging as critical routes for the epitranscriptomic control of gene expression

RNA Controls PolyQ Protein Phase Transitions

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information, but also the biophysical properties of phase-separated compartments