Novel mRNA-containing cytoplasmic granules in ALK-transformed cells

In mammalian cells, nontranslating messenger RNAs (mRNAs) are concentrated in different cytoplasmic foci, such as processing bodies (PBs) and stress granules (SGs), where they are either degraded or stored. In the present study, we have thoroughly characterized cytoplasmic foci, hereafter called AGs for ALK granules that form in transformed cells expressing the constitutively active anaplastic lymphoma kinase (ALK). AGs contain polyadenylated mRNAs and a unique combination of several RNA binding proteins that so far has not been described in mammalian foci, including AUF1, HuR, and the poly (A(+)) binding protein PABP. AGs shelter neither components of the mRNA degradation machinery present in PBs nor known markers of SGs, such as translation initiation factors or TIA/TIAR, showing that they are distinct from PBs or SGs. AGs and PBs, however, both move on microtubules with similar dynamics and frequently establish close contacts. In addition, in conditions in which mRNA metabolism is perturbed, AGs concentrate PB components with the noticeable exception of the 5' to 3' exonuclease XRN1. Altogether, we show that AGs constitute novel mRNA-containing cytoplasmic foci and we propose that they could protect translatable mRNAs from degradation, contributing thus to ALK-mediated oncogenicity.     

Plant stress granules and mRNA processing bodies are distinct from heat stress granules

Similar to the situation in mammalian cells and yeast, messenger ribonucleo protein (mRNP) homeostasis in plant cells depends on rapid transitions between three functional states, i.e. translated mRNPs in polysomes, stored mRNPs and mRNPs under degradation. Studies in mammalian cells showed that whenever the dynamic exchange of the components between these states is disrupted, stalled mRNPs accumulate in cytoplasmic aggregates, such as stress granules (SGs) or processing bodies (PBs). We identified PBs and SGs in plant cells by detection of DCP1, DCP2 and XRN4, as marker proteins for the 5'-->3' mRNA degradation pathway, and eIF4E, as well as the RNA binding proteins RBP47 and UBP1, as marker proteins for stored mRNPs in SGs. Cycloheximide-inhibited translation, stress treatments and mutants defective in mRNP homeostasis were used to study the dynamic transitions of mRNPs between SGs and PBs. SGs and PBs can be clearly discriminated from the previously described heat stress granules (HSGs), which evidently do not contain mRNPs. Thus, the role of HSGs as putative mRNP storage sites must be revised.     

Yeast processing bodies and stress granules: self-assembly ribonucleoprotein particles

Processing bodies (PBs) and stress granules (SGs) are two highly conserved cytoplasmic ribonucleoprotein foci that contain translationally repressed mRNAs together with proteins from the mRNA metabolism. Interestingly, they also share some common features with other granules, including the prokaryotic inclusion bodies. Although the function of PBs and SGs remains elusive, major advances have been done in unraveling their composition and assembly by using the yeast Saccharomyces cerevisae.     

Hsp90 Regulates the Function of Argonaute 2 and Its Recruitment to Stress Granules and P-Bodies

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNA-activated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs.     

The involvement of stress granules in aging and aging‐associated diseases

Stress granules (SGs) are nonmembrane assemblies formed in cells in response to stress conditions. SGs mainly contain untranslated mRNA and a variety of proteins. RNAs and scaffold proteins with intrinsically disordered regions or RNA‐binding domains are essential for the assembly of SGs, and multivalent macromolecular interactions among these components are thought to be the driving forces for SG assembly. The SG assembly process includes regulation through post‐translational modification and involvement of the cytoskeletal system. During aging, many intracellular bioprocesses become disrupted by factors such as cellular environmental changes, mitochondrial dysfunction, and decline in the protein quality control system. Such changes could lead to the formation of aberrant SGs, as well as alterations in their maintenance, disassembly, and clearance. These aberrant SGs might in turn promote aging and aging‐associated diseases. In this paper, we first review the latest progress on the molecular mechanisms underlying SG assembly and SG functioning under stress conditions. Then, we provide a detailed discussion of the relevance of SGs to aging and aging‐associated diseases.     

BUHO: A MATLAB Script for the Study of Stress Granules and Processing Bodies by High-Throughput Image Analysis

The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny.     

Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO2), cycloheximide (CHX) and Lipofectamine 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi. Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO2. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.     

RNA is required for the integrity of multiple nuclear and cytoplasmic membrane‐less RNP granules

Numerous membrane‐less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super‐enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane‐less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.     

ZBP1 regulates mRNA stability during cellular stress

An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein-mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.     

Perspective on architecture and assembly of membrane contact sites

Membrane contact sites (MCS) are platforms of physical contact between different organelles. They are formed through interactions involving lipids and proteins, and function in processes such as calcium and lipid exchange, metabolism and organelle biogenesis. In this article, we discuss emerging questions regarding the architecture, organisation and assembly of MCS, such as: What is the contribution of different components to the interaction between organelles? How is the specific composition of different types of membrane contacts sites established and maintained? How are proteins and lipids spatially organised at MCS and how does that influence their function? How dynamic are MCS on the molecular and ultrastructural level? We highlight current state of research and point out experimental approaches that promise to contribute to a spatiomechanistic understanding of MCS functions.     

Induction of Stress Granule-Like Structures in Vesicular Stomatitis Virus-Infected Cells

Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.     

Analysis of stress granule assembly in Schizosaccharomyces pombe

Stress granules (SGs) are cytoplasmic aggregates of RNA and proteins in eukaryotic cells that are rapidly induced in response to environmental stress, but are not seen in cells growing under favorable conditions. SGs have been primarily studied in mammalian cells. The existence of SGs in the fission yeast and the distantly related budding yeast was demonstrated only recently. In both species, they contain many orthologs of the proteins seen in mammalian SGs. In this study, we have characterized these proteins and determined their involvement in the assembly of fission yeast SGs, in particular, the homolog of human G3BP proteins. G3BP interacts with the deubiquitinating protease USP10 and plays an important role in the assembly of SGs. We have also identified Ubp3, an ortholog of USP10, as an interaction partner of the fission yeast G3BP-like protein Nxt3 and required for its stability. Under thermal stress, like their human orthologs, both Nxt3 and Ubp3 rapidly relocalize to cytoplasmic foci that contain the SG marker poly(A)-binding protein Pabp. However, in contrast to G3BP1 and USP10, neither deletion nor overexpression of nxt3(+) or ubp3(+) affected the assembly of fission yeast SGs as judged by the relocalization of Pabp. Similar results were observed in mutants defective in orthologs of SG components that are known to affect SG assembly in human and in budding yeast, such as ataxia-2 and TIA-like proteins. Together, our data indicate that despite similar protein compositions, the underlying molecular mechanisms for the assembly of SGs could be distinct between species.     

The dynamics of mammalian P body transport, assembly, and disassembly in vivo

Exported mRNAs are targeted for translation or can undergo degradation by several decay mechanisms. The 5′→3′ degradation machinery localizes to cytoplasmic P bodies (PBs). We followed the dynamic properties of PBs in vivo and investigated the mechanism by which PBs scan the cytoplasm. Using proteins of the decapping machinery, we asked whether PBs actively scan the cytoplasm or whether a diffusion-based mechanism is sufficient. Live-cell imaging showed that PBs were anchored mainly to microtubules. Quantitative single-particle tracking demonstrated that most PBs exhibited spatially confined motion dependent on microtubule motion, whereas stationary PB pairs were identified at the centrosome. Some PBs translocated in long-range movements on microtubules. PB mobility was compared with mitochondria, endoplasmic reticulum, peroxisomes, SMN bodies, and stress granules, and diffusion coefficients were calculated. Disruption of the microtubule network caused a significant reduction in PB mobility together with an induction of PB assembly. However, FRAP measurements showed that the dynamic flux of assembled PB components was not affected by such treatments. FRAP analysis showed that the decapping enzyme Dcp2 is a nondynamic PB core protein, whereas Dcp1 proteins continuously exchanged with the cytoplasm. This study reveals the mechanism of PB transport, and it demonstrates how PB assembly and disassembly integrate with the presence of an intact cytoskeleton.     

Stress granules,RNA-binding proteins and polyglutamine diseases: too much aggregation?

Stress granules (SGs) are membraneless cell compartments formed in response to different stress stimuli, wherein translation factors, mRNAs, RNA-binding proteins (RBPs) and other proteins coalesce together. SGs assembly is crucial for cell survival, since SGs are implicated in the regulation of translation, mRNA storage and stabilization and cell signalling, during stress. One defining feature of SGs is their dynamism, as they are quickly assembled upon stress and then rapidly dispersed after the stress source is no longer present. Recently, SGs dynamics, their components and their functions have begun to be studied in the context of human diseases. Interestingly, the regulated protein self-assembly that mediates SG formation contrasts with the pathological protein aggregation that is a feature of several neurodegenerative diseases. In particular, aberrant protein coalescence is a key feature of polyglutamine (PolyQ) diseases, a group of nine disorders that are caused by an abnormal expansion of PolyQ tract-bearing proteins, which increases the propensity of those proteins to aggregate. Available data concerning the abnormal properties of the mutant PolyQ disease-causing proteins and their involvement in stress response dysregulation strongly suggests an important role for SGs in the pathogenesis of PolyQ disorders. This review aims at discussing the evidence supporting the existence of a link between SGs functionality and PolyQ disorders, by focusing on the biology of SGs and on the way it can be altered in a PolyQ disease context.Subject terms: Neuroscience, Neurological disorders