The Parkinson's disease protein alpha-synuclein is a modulator of processing bodies and mRNA stability.

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.

m6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3.

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

Dynamic "Cap"-abilities of P-bodies and the XRN1-EDC4 axis.

RNA turnover regulates the quality and quantity of cellular gene expression through a coordinated cavalcade of enzymes, factors, and phase transitions. In this issue, Brothers et al reveal the importance of balanced communication between the Xrn1 exonuclease and the EDC4 decapping factor to coordinate P-body dynamics and maintain cellular fitness.

An actin remodeling role for Arabidopsis processing bodies revealed by their proximity interactome.

Cellular condensates can comprise membrane-less ribonucleoprotein assemblies with liquid-like properties. These cellular condensates influence various biological outcomes, but their liquidity hampers their isolation and characterization. Here, we investigated the composition of the condensates known as processing bodies (PBs) in the model plant Arabidopsis thaliana through a proximity-biotinylation proteomics approach. Using in situ protein-protein interaction approaches, genetics and high-resolution dynamic imaging, we show that processing bodies comprise networks that interface with membranes. Surprisingly, the conserved component of PBs, DECAPPING PROTEIN 1 (DCP1), can localize to unique plasma membrane subdomains including cell edges and vertices. We characterized these plasma membrane interfaces and discovered a developmental module that can control cell shape. This module is regulated by DCP1, independently from its role in decapping, and the actin-nucleating SCAR-WAVE complex, whereby the DCP1-SCAR-WAVE interaction confines and enhances actin nucleation. This study reveals an unexpected function for a conserved condensate at unique membrane interfaces.

The EDC4-XRN1 interaction controls P-body dynamics to link mRNA decapping with decay.

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'-3' exonuclease XRN1 interact with the enhancer of mRNA-decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4-XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P-bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5'-3' decay in human cells.

A proxitome-RNA-capture approach reveals that processing bodies repress coregulated hub genes.

Cellular condensates are usually ribonucleoprotein assemblies with liquid- or solid-like properties. Because these subcellular structures lack a delineating membrane, determining their compositions is difficult. Here we describe a proximity-biotinylation approach for capturing the RNAs of the condensates known as processing bodies (PBs) in Arabidopsis (Arabidopsis thaliana). By combining this approach with RNA detection, in silico, and high-resolution imaging approaches, we studied PBs under normal conditions and heat stress. PBs showed a much more dynamic RNA composition than the total transcriptome. RNAs involved in cell wall development and regeneration, plant hormonal signaling, secondary metabolism/defense, and RNA metabolism were enriched in PBs. RNA-binding proteins and the liquidity of PBs modulated RNA recruitment, while RNAs were frequently recruited together with their encoded proteins. In PBs, RNAs follow distinct fates: in small liquid-like PBs, RNAs get degraded while in more solid-like larger ones, they are stored. PB properties can be regulated by the actin-polymerizing SCAR (suppressor of the cyclic AMP)-WAVE (WASP family verprolin homologous) complex. SCAR/WAVE modulates the shuttling of RNAs between PBs and the translational machinery, thereby adjusting ethylene signaling. In summary, we provide an approach to identify RNAs in condensates that allowed us to reveal a mechanism for regulating RNA fate.

Stress granule and P-body clearance: Seeking coherence in acts of disappearance.

Stress granules and P-bodies are conserved cytoplasmic biomolecular condensates whose assembly and composition are well documented, but whose clearance mechanisms remain controversial or poorly described. Such understanding could provide new insight into how cells regulate biomolecular condensate formation and function, and identify therapeutic strategies in disease states where aberrant persistence of stress granules in particular is implicated. Here, I review and compare the contributions of chaperones, the cytoskeleton, post-translational modifications, RNA helicases, granulophagy and the proteasome to stress granule and P-body clearance. Additionally, I highlight the potentially vital role of RNA regulation, cellular energy, and changes in the interaction networks of stress granules and P-bodies as means of eliciting clearance. Finally, I discuss evidence for interplay of distinct clearance mechanisms, suggest future experimental directions, and suggest a simple working model of stress granule clearance.

Composition and function of stress granules and P-bodies in plants.

Stress Granules (SGs) and Processing-bodies (P-bodies) are biomolecular condensates formed in the cell with the highly conserved purpose of maintaining balance between storage, translation, and degradation of mRNA. This balance is particularly important when cells are exposed to different environmental conditions and adjustments have to be made in order for plants to respond to and tolerate stressful conditions. While P-bodies are constitutively present in the cell, SG formation is a stress-induced event. Typically thought of as protein-RNA aggregates, SGs and P-bodies are formed by a process called liquid-liquid phase separation (LLPS), and both their function and composition are very dynamic. Both foci are known to contain proteins involved in translation, protein folding, and ATPase activity, alluding to their roles in regulating mRNA and protein expression levels. From an RNA perspective, SGs and P-bodies primarily consist of mRNAs, though long non-coding RNAs (lncRNAs) have also been observed, and more focus is now being placed on the specific RNAs associated with these aggregates. Recently, metabolites such as nucleotides and amino acids have been reported in purified plant SGs with implications for the energetic dynamics of these condensates. Thus, even though the field of plant SGs and P-bodies is relatively nascent, significant progress has been made in understanding their composition and biological role in stress responses. In this review, we discuss the most recent discoveries centered around SG and P-body function and composition in plants.

P-body-like condensates in the germline.

P-bodies are cytoplasmic condensates that accumulate low-translation mRNAs for temporary storage before translation or degradation. P-bodies have been best characterized in yeast and mammalian tissue culture cells. We describe here related condensates in the germline of animal models. Germline P-bodies have been reported at all stages of germline development from primordial germ cells to gametes. The activity of the universal germ cell fate regulator, Nanos, is linked to the mRNA decay function of P-bodies, and spatially-regulated condensation of P-body like condensates in embryos is required to localize mRNA regulators to primordial germ cells. In most cases, however, it is not known whether P-bodies represent functional compartments or non-functional condensation by-products that arise when ribonucleoprotein complexes saturate the cytoplasm. We speculate that the ubiquity of P-body-like condensates in germ cells reflects the strong reliance of the germline on cytoplasmic, rather than nuclear, mechanisms of gene regulation.

The P-body component DECAPPING5 and the floral repressor SISTER OF FCA regulate FLOWERING LOCUS C transcription in Arabidopsis.

Flowering is the transition from vegetative to reproductive growth and is critical for plant adaptation and reproduction. FLOWERING LOCUS C (FLC) plays a central role in flowering time control, and dissecting its regulation mechanism provides essential information for crop improvement. Here, we report that DECAPPING5 (DCP5), a component of processing bodies (P-bodies), regulates FLC transcription and flowering time in Arabidopsis (Arabidopsis thaliana). DCP5 and its interacting partner SISTER OF FCA (SSF) undergo liquid-liquid phase separation (LLPS) that is mediated by their prion-like domains (PrDs). Enhancing or attenuating the LLPS of both proteins using transgenic methods greatly affects their ability to regulate FLC and flowering time. DCP5 regulates FLC transcription by modulating RNA polymerase II enrichment at the FLC locus. DCP5 requires SSF for FLC regulation, and loss of SSF or its PrD disrupts DCP5 function. Our results reveal that DCP5 interacts with SSF, and the nuclear DCP5-SSF complex regulates FLC expression at the transcriptional level.

Combined modelling of mRNA decay dynamics and single-molecule imaging in the Drosophila embryo uncovers a role for P-bodies in 5' to 3' degradation.

Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.

Quantitative reconstitution of yeast RNA processing bodies.

Many biomolecular condensates appear to form through liquid-liquid phase separation (LLPS). Individual condensate components can often undergo LLPS in vitro, capturing some features of the native structures. However, natural condensates contain dozens of components with different concentrations, dynamics, and contributions to compartment formation. Most biochemical reconstitutions of condensates have not benefited from quantitative knowledge of these cellular features nor attempted to capture natural complexity. Here, we build on prior quantitative cellular studies to reconstitute yeast RNA processing bodies (P bodies) from purified components. Individually, five of the seven highly concentrated P-body proteins form homotypic condensates at cellular protein and salt concentrations, using both structured domains and intrinsically disordered regions. Combining the seven proteins together at their cellular concentrations with RNA yields phase-separated droplets with partition coefficients and dynamics of most proteins in reasonable agreement with cellular values. RNA delays the maturation of proteins within and promotes the reversibility of, P bodies. Our ability to quantitatively recapitulate the composition and dynamics of a condensate from its most concentrated components suggests that simple interactions between these components carry much of the information that defines the physical properties of the cellular structure.

Specialized germline P-bodies are required to specify germ cell fate in Caenorhabditis elegans embryos.

In animals with germ plasm, specification of the germline involves 'germ granules', cytoplasmic condensates that enrich maternal transcripts in the germline founder cells. In Caenorhabditis elegans embryos, P granules enrich maternal transcripts, but surprisingly P granules are not essential for germ cell fate specification. Here, we describe a second condensate in the C. elegans germ plasm. Like canonical P-bodies found in somatic cells, 'germline P-bodies' contain regulators of mRNA decapping and deadenylation and, in addition, the intrinsically-disordered proteins MEG-1 and MEG-2 and the TIS11-family RNA-binding protein POS-1. Embryos lacking meg-1 and meg-2 do not stabilize P-body components, misregulate POS-1 targets, mis-specify the germline founder cell and do not develop a germline. Our findings suggest that specification of the germ line involves at least two distinct condensates that independently enrich and regulate maternal mRNAs in the germline founder cells. This article has an associated 'The people behind the papers' interview.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi's sarcoma (KS). We previously demonstrated that the KSHV Kaposin B (KapB) protein promotes inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). PBs modify gene expression by silencing or degrading labile messenger RNAs (mRNAs), including many transcripts that encode inflammatory or angiogenic proteins associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via phosphorylation of the autophagy regulatory protein, Beclin. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin phosphorylation or autophagic flux. Moreover, we showed that PB disassembly mediated by KSHV or KapB, depended on autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

Arabidopsis RNA processing body components LSM1 and DCP5 aid in the evasion of translational repression during Cauliflower mosaic virus infection.

Viral infections impose extraordinary RNA stress, triggering cellular RNA surveillance pathways such as RNA decapping, nonsense-mediated decay, and RNA silencing. Viruses need to maneuver among these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells, with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigated the role of Arabidopsis thaliana PBs during Cauliflower mosaic virus (CaMV) infection. We found that several PB components are co-opted into viral factories that support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we established that PB components are helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, dysfunctions in PB components expose the virus to this pathway, which is similar to previous observations for transgenes. Transgenes, however, undergo RNA quality control-dependent RNA degradation and transcriptional silencing, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence among PBs, RNA silencing, and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.

RNA helicase DDX6 and scaffold protein GW182 in P-bodies promote biogenesis of stress granules.

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies.

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs are not affected. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.

Stress-granules, P-bodies, and cell aging: A bioinformatics study.

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30-40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SGs proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.

In vivo proximity biotin ligation identifies the interactome of Egalitarian, a Dynein cargo adaptor.

Numerous motors of the Kinesin family contribute to plus-end-directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In selected cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. We used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be linked to Dynein in an Egalitarian-dependent manner. We therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. We also devised and validated a nanobody-based proximity biotinylation strategy that can be used to define the interactome of any GFP-tagged protein.

What are the distinguishing features and size requirements of biomolecular condensates and their implications for RNA-containing condensates?

Exciting recent work has highlighted that numerous cellular compartments lack encapsulating lipid bilayers (often called "membraneless organelles"), and that their structure and function are central to the regulation of key biological processes, including transcription, RNA splicing, translation, and more. These structures have been described as "biomolecular condensates" to underscore that biomolecules can be significantly concentrated in them. Many condensates, including RNA granules and processing bodies, are enriched in proteins and nucleic acids. Biomolecular condensates exhibit a range of material states from liquid- to gel-like, with the physical process of liquid-liquid phase separation implicated in driving or contributing to their formation. To date, in vitro studies of phase separation have provided mechanistic insights into the formation and function of condensates. However, the link between the often micron-sized in vitro condensates with nanometer-sized cellular correlates has not been well established. Consequently, questions have arisen as to whether cellular structures below the optical resolution limit can be considered biomolecular condensates. Similarly, the distinction between condensates and discrete dynamic hub complexes is debated. Here we discuss the key features that define biomolecular condensates to help understand behaviors of structures containing and generating RNA.