Control of RNA Stability in Immunity.

Posttranscriptional control of mRNA regulates various biological processes, including inflammatory and immune responses. RNA-binding proteins (RBPs) bind cis-regulatory elements in the 3' untranslated regions (UTRs) of mRNA and regulate mRNA turnover and translation. In particular, eight RBPs (TTP, AUF1, KSRP, TIA-1/TIAR, Roquin, Regnase, HuR, and Arid5a) have been extensively studied and are key posttranscriptional regulators of inflammation and immune responses. These RBPs sometimes collaboratively or competitively bind the same target mRNA to enhance or dampen regulatory activities. These RBPs can also bind their own 3' UTRs to negatively or positively regulate their expression. Both upstream signaling pathways and microRNA regulation shape the interactions between RBPs and target RNA. Dysregulation of RBPs results in chronic inflammation and autoimmunity. Here, we summarize the functional roles of these eight RBPs in immunity and their associated diseases.

Comparison of protection against mpox following mRNA or modified vaccinia Ankara vaccination in nonhuman primates.

In 2022, mpox virus (MPXV) spread worldwide, causing 99,581 mpox cases in 121 countries. Modified vaccinia Ankara (MVA) vaccine use reduced disease in at-risk populations but failed to deliver complete protection. Lag in manufacturing and distribution of MVA resulted in additional MPXV spread, with 12,000 reported cases in 2023 and an additional outbreak in Central Africa of clade I virus. These outbreaks highlight the threat of zoonotic spillover by Orthopoxviruses. mRNA-1769, an mRNA-lipid nanoparticle (LNP) vaccine expressing MPXV surface proteins, was tested in a lethal MPXV primate model. Similar to MVA, mRNA-1769 conferred protection against challenge and further mitigated symptoms and disease duration. Antibody profiling revealed a collaborative role between neutralizing and Fc-functional extracellular virion (EV)-specific antibodies in viral restriction and ospinophagocytic and cytotoxic antibody functions in protection against lesions. mRNA-1769 enhanced viral control and disease attenuation compared with MVA, highlighting the potential for mRNA vaccines to mitigate future pandemic threats.

A multivalent mRNA monkeypox virus vaccine (BNT166) protects mice and macaques from orthopoxvirus disease.

In response to the 2022 outbreak of mpox driven by unprecedented human-to-human monkeypox virus (MPXV) transmission, we designed BNT166, aiming to create a highly immunogenic, safe, accessible, and scalable next-generation vaccine against MPXV and related orthopoxviruses. To address the multiple viral forms and increase the breadth of immune response, two candidate multivalent mRNA vaccines were evaluated pre-clinically: a quadrivalent vaccine (BNT166a; encoding the MPXV antigens A35, B6, M1, H3) and a trivalent vaccine (BNT166c; without H3). Both candidates induced robust T cell responses and IgG antibodies in mice, including neutralizing antibodies to both MPXV and vaccinia virus. In challenge studies, BNT166a and BNT166c provided complete protection from vaccinia, clade I, and clade IIb MPXV. Furthermore, immunization with BNT166a was 100% effective at preventing death and at suppressing lesions in a lethal clade I MPXV challenge in cynomolgus macaques. These findings support the clinical evaluation of BNT166, now underway (NCT05988203).

RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

The FXR1 network acts as a signaling scaffold for actomyosin remodeling.

It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the fragile X-related protein 1 (FXR1) network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as an underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. Our findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as an organizer of signaling reactions.

The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Translation and mRNA Stability Control.

Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cis-regulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.

3'-End Processing of Eukaryotic mRNA: Machinery, Regulation, and Impact on Gene Expression.

Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.

ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines.

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared with conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for 3 months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

Self-demixing of mRNA copies buffers mRNA:mRNA and mRNA:regulator stoichiometries.

Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection.

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).

Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants.

The BQ and XBB subvariants of SARS-CoV-2 Omicron are now rapidly expanding, possibly due to altered antibody evasion properties deriving from their additional spike mutations. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals boosted with a WA1/BA.5 bivalent mRNA vaccine. Titers against BQ and XBB subvariants were lower by 13- to 81-fold and 66- to 155-fold, respectively, far beyond what had been observed to date. Monoclonal antibodies capable of neutralizing the original Omicron variant were largely inactive against these new subvariants, and the responsible individual spike mutations were identified. These subvariants were found to have similar ACE2-binding affinities as their predecessors. Together, our findings indicate that BQ and XBB subvariants present serious threats to current COVID-19 vaccines, render inactive all authorized antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.

Sites of transcription initiation drive mRNA isoform selection.

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.

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.

Boosting with variant-matched or historical mRNA vaccines protects against Omicron infection in mice.

The large number of spike substitutions in Omicron lineage variants (BA.1, BA.1.1., and BA.2) could jeopardize the efficacy of SARS-CoV-2 vaccines. We evaluated in mice the protective efficacy of the Moderna mRNA-1273 vaccine against BA.1 before or after boosting. Whereas two doses of mRNA-1273 vaccine induced high levels of neutralizing antibodies against historical WA1/2020 strains, lower levels against BA.1 were associated with breakthrough infection and inflammation in the lungs. A primary vaccination series with mRNA-1273.529, an Omicron-matched vaccine, potently neutralized BA.1 but inhibited historical or other SARS-CoV-2 variants less effectively. However, boosting with either mRNA-1273 or mRNA-1273.529 vaccines increased neutralizing titers and protection against BA.1 and BA.2 infection. Nonetheless, the neutralizing antibody titers were higher, and lung viral burden and cytokines were slightly lower in mice boosted with mRNA-1273.529 and challenged with BA.1. Thus, boosting with mRNA-1273 or mRNA-1273.529 enhances protection against Omicron infection with limited differences in efficacy measured.

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung.

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log10, and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

Interval between prior SARS-CoV-2 infection and booster vaccination impacts magnitude and quality of antibody and B cell responses.

SARS-CoV-2 mRNA booster vaccines provide protection from severe disease, eliciting strong immunity that is further boosted by previous infection. However, it is unclear whether these immune responses are affected by the interval between infection and vaccination. Over a 2-month period, we evaluated antibody and B cell responses to a third-dose mRNA vaccine in 66 individuals with different infection histories. Uninfected and post-boost but not previously infected individuals mounted robust ancestral and variant spike-binding and neutralizing antibodies and memory B cells. Spike-specific B cell responses from recent infection (<180 days) were elevated at pre-boost but comparatively less so at 60 days post-boost compared with uninfected individuals, and these differences were linked to baseline frequencies of CD27lo B cells. Day 60 to baseline ratio of BCR signaling measured by phosphorylation of Syk was inversely correlated to days between infection and vaccination. Thus, B cell responses to booster vaccines are impeded by recent infection.