Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function

The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3’UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3’UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.

First person – Akruti Shah

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Akruti Shah is first author on ‘ IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap’, published in JCS. Akruti is a PhD student in the lab of Dr Rashna Bhandari at the Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, India, investigating the effect of post-translational modifications and protein–protein interactions on mRNA metabolism in health and disease.

Processing body dynamics drive non-genetic MEK inhibitors tolerance by fine-tuning KRAS and NRAS translation

BackgroundOveractivation of the Mitogen-activated protein kinase (MAPK) pathway is a critical driver of many human cancers. However, therapies that target this pathway have only been effective in a few cancers, as cancers inevitably end up developing resistance. Puzzling observations have suggested that MAPK targeting in tumor fails because of an early compensatory RAS overexpression, but through unexplained mechanisms.MethodsLung, breast, and melanoma cancer cells were incubated with MEK inhibitors (MEKi). Kinetics of expression of KRAS, NRAS mRNA and proteins and processing bodies (PBs) proteins were followed overtime by immunoblot and confocal studies.ResultsHere, we identified a novel mechanism of drug tolerance for MEKi involving PBs essential proteins like DDX6 or LSM14A. MEKi promoted the translation of KRAS and NRAS oncogenes, which in turn triggered BRAF phosphorylation. This overexpression, which occurred in the absence of neo-transcription, depended on PBs dissolution as a source of RAS mRNA reservoir. In addition, in response to MEKi removal, we showed that the process was dynamic since the PBs quickly reformed, reducing MAPK signaling. These results underline a dynamic spatiotemporal negative feedback loop of MAPK signaling via RAS mRNA sequestration. Furthermore, in long-tolerant cells, we observed a LSM14A loss of expression that promoted a low PBs number phenotype together with strong KRAS and NRAS induction capacities.ConclusionsAltogether we describe here a new intricate mechanism involving PB, DDX6 and LSM14A in the translation regulation of essential cellular pathways that pave the way for future therapies altering PBs dissolution to improve cancer targeted-drug therapies.

IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap

Inositol hexakisphosphate kinase 1 (IP6K1) is a small molecule kinase that catalyzes the conversion of the inositol phosphate IP6 to 5-IP7. We show that IP6K1 acts independent of its catalytic activity to upregulate the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that store translationally repressed mRNA. IP6K1 does not localize to P-bodies, but instead binds to ribosomes, where it interacts with the mRNA decapping complex - the scaffold protein EDC4, activator proteins DCP1A/B, decapping enzyme DCP2, and RNA helicase DDX6. Along with its partner 4E-T, DDX6 is known to nucleate protein-protein interactions on the 5’ mRNA cap to facilitate P-body formation. IP6K1 binds the translation initiation complex eIF4F on the mRNA cap, augmenting the interaction of DDX6 with 4E-T and the cap binding protein eIF4E. Cells with reduced IP6K1 show downregulated microRNA-mediated translational suppression and increased stability of DCP2-regulated transcripts. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translational repression over initiation, thus leading to P-body assembly.

Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

Using quantitative reconstitution to investigate multi-component condensates

Many biomolecular condensates are thought to form via liquid-liquid phase separation (LLPS) of multivalent macromolecules. For those that form through this mechanism, our understanding has benefitted significantly from biochemical reconstitutions of key components and activities. Reconstitutions of RNA-based condensates to date have mostly been based on relatively simple collections of molecules. However, proteomics and sequencing data indicate that natural RNA-based condensates are enriched in hundreds to thousands of different components, and genetic data suggest multiple interactions can contribute to condensate formation to varying degrees. In this perspective we describe recent progress in understanding RNA-based condensates through different levels of biochemical reconstitutions, as a means to bridge the gap between simple in vitro reconstitution and cellular analyses. Complex reconstitutions provide insight into the formation, regulation, and functions of multi-component condensates. We focus on two RNA-protein condensate case studies: stress granules and RNA processing bodies (P bodies), and examine the evidence for cooperative interactions among multiple components promoting LLPS. An important concept emerging from these studies is that composition and stoichiometry regulate biochemical activities within condensates. Based on the lessons learned from stress granules and P bodies we discuss forward-looking approaches to understand the thermodynamic relationships between condensate components, with the goal of developing predictive models of composition and material properties, and their effects on biochemical activities. We anticipate that quantitative reconstitutions will facilitate understanding of the complex thermodynamics and functions of diverse RNA-protein condensates.

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.

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Influence of Feedstock and Final Pyrolysis Temperature on Breaking Strength and Dust Production of Wood-Derived Biochars

The susceptibility to fragmentation of biochar is an important property to consider in field applications. Physical and mechanical properties of wood-derived biochars from vine shoots and holm oak were studied to evaluate the effect of biomass feedstock, final pyrolysis temperature and application conditions. Vine shoots and holm oak pruning residues were selected for biochar production. Slow pyrolysis experiments were conducted at two different final temperatures (400 and 600 °C). Physical and chemical characteristics of biomass and biochars were determined. Impact strength was evaluated through the measurement of the gravitational potential energy per unit area (J mm−2) necessary for the breakage of biochar fragments. Shear strength (N mm−2) and a combination of shear/compression strengths (N) were analyzed using a Universal Texture Analyzer. A particular mechanical treatment was carried out on biochar samples to simulate the processing bodies of a commercial manure spreader, under two gravimetric moisture contents. Holm oak-derived biochar was more resistant than vine shoot-derived biochar to the applied forces. Vine shoots-derived biochar did not show a significantly different mechanical behavior between temperatures. Holm Oak-derived biochar produced at the higher final pyrolysis temperature showed higher resistance to be broken into smaller pieces. Moistening resulted in an adequate practice to improve mechanical spreading.

RNA helicase DDX6 in P-bodies is essential for the assembly of stress granules

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate storage of translationally arrested mRNAs and are intimately related. In this study, we found the dependence of SG formation on PB in the cells under arsenite (ARS) stress, but not the other way around. GW182, 4E-T and DDX6 essential for PB formation differentially affect SG formation in the cells under ARS stress, with DDX6 being the most prominent. The cells with DDX6 deficiency display irregular shape of SG which could be rescued by ectopic wt DDX6, but not its helicase mutant E247A DDX6, which induces SG in the cells without stress, indicating that DDX6 helicase activity is essential for PB, but suppressive for SG. DDX6's dual roles are independent of DDX6 interactors EDC3, CNOT1, and PAT1B. This study provides a conceptual advance of how DDX6 involves in the biogenesis of PB and SG.