Alessia Ruggieri

Stress granules (SGs) are biomolecular condensates that form transiently in the cytosol of mammalian cells in response to stress. Dysregulation of their assembly or disassembly is implicated in human age-related diseases. While phase separation is the key process underlying SG assembly, understanding of their function, composition and regulation in response to physiological stimuli is limited. This knowledge gap reflects the challenge of gaining comprehensive and quantitative insights into the dynamic regulation of the complex composition of SGs at the single-cell level. Here we present an emulsion-based microfluidics method to overcome this limitation. “Cytosolic extracts-in-oil droplets” (CEODs) recreate a confined active cytosolic milieu that undergoes phase separation and SG formation in response to stress under physiological conditions. This approach led to the discovery of seven previously unrecognised SG components involved in signalling pathways. CEODs provide a versatile and cost-effective screening platform for future mechanistic and therapeutic studies.

Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes, including RNA stability and translation. Although advances in analytical methods have improved the detection of RNA modifications, precise mapping at single-base resolution remains challenging. Requirements for sensitivity and purity limit accuracy and reproducibility, especially for low abundant viral RNAs extracted from infected cells. Here, we report a two-step method, ViREn, for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a single high-confidence 5-methylcytosine (m5C) site in DENV gRNA at position 1218. This finding was orthogonally validated by Illumina-based bisulfite sequencing and by Nanopore Oxford Technologies direct RNA sequencing. Strikingly, m5C1218 was detected exclusively in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m5C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m5C on low-abundance viral gRNA with unprecedented precision and sensitivity and facilitates future mechanistic studies into the role of RNA modifications in virus replication.

Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes including RNA stability and translation. Although advances in analytical methods have improved detection sensitivity, the precise mapping of RNA modifications at single-base resolution remains challenging. Especially for low abundant viral RNAs extracted from infected cells, requirements for sensitivity and purity limit accuracy and reproducibility. Here we report the two-step method ViREn for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a high-confidence 5-methylcytosine (m ⁵ C) site in DENV gRNA, orthogonally validated by Illumina-based bisulfite sequencing and direct RNA sequencing by Nanopore Oxford Technologies. Strikingly, this m ⁵ C modification was exclusively detected in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m ⁵ C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m ⁵ C on low abundance viral gRNA with unprecedented precision and sensitivity and facilitates mechanistic studies into the role of RNA modification in virus replication.

Summary: The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of Growth Arrest and DNA Damage inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We established that the 3’ untranslated region of PPP1R15A mRNA contains an active AU-rich element recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a key step in establishing the ISR molecular memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.

The cellular integrated stress response (ISR) is a central signaling pathway that tunes translation initiation in response to a wide range of cellular insults to promote cell survival. The critical node of this regulation involves the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) by stress kinases. In this issue of EMBO reports, Wu et al (2023) report FAM69C as a novel eIF2α kinase that promotes ISR activation and stress granule (SG) assembly in microglia in response to oxidative stress. This work proposes a protective role for FAM69C and SGs in limiting damaging inflammatory responses commonly associated with neurodegenerative diseases.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage^1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response, by integrating quantitative experiments with mathematical modeling, and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 mRNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

The integrated stress response is a highly regulated signaling cascade that allows cells to react to a variety of external and internal stimuli. Activation of different stress-responsive kinases leads to the phosphorylation of their common downstream target, the eukaryotic translation initiation factor 2 alpha (eIF2α), which is a critical component of functional translation preinitiation complexes. As a consequence, stalled ribonucleoprotein complexes accumulate in the cytoplasm and condense into microscopically visible cytoplasmic stress granules (SGs). Over the past years, numerous microscopy approaches have been developed to study the spatiotemporal control of SG formation in response to a variety of stressors. Here, we apply long-term live-cell microscopy to monitor the dynamic cellular stress response triggered by infection with chronic hepatitis C virus (HCV) at single-cell level and study the behavior of infected cells that repeatedly switch between a stressed and unstressed state. We describe in detail the engineering of fluorescent SG-reporter cells expressing enhanced yellow fluorescent protein (YFP)-tagged T cell internal antigen 1 (TIA-1) using lentiviral delivery, as well as the production of mCherry-tagged HCV trans-complemented particles, which allow live tracking of SG assembly and disassembly, SG number and size in single infected cells over time.