Shiftless Is a Novel Member of the Ribosome Stress Surveillance Machinery That Has Evolved to Play a Role in Innate Immunity and Cancer Surveillance.

A longstanding paradox in molecular biology has centered on the question of how very long proteins are synthesized, despite numerous measurements indicating that ribosomes spontaneously shift reading frame at rates that should preclude their ability completely translate their mRNAs. Shiftless (SFL; C19orf66) was originally identified as an interferon responsive gene encoding an antiviral protein, indicating that it is part of the innate immune response. This activity is due to its ability to bind ribosomes that have been programmed by viral sequence elements to shift reading frame. Curiously, Shiftless is constitutively expressed at low levels in mammalian cells. This study examines the effects of altering Shiftless homeostasis, revealing how it may be used by higher eukaryotes to identify and remove spontaneously frameshifted ribosomes, resolving the apparent limitation on protein length. Data also indicate that Shiftless plays a novel role in the ribosome-associated quality control program. A model is proposed wherein SFL recognizes and arrests frameshifted ribosomes, and depending on SFL protein concentrations, either leads to removal of frameshifted ribosomes while leaving mRNAs intact, or to mRNA degradation. We propose that SFL be added to the growing pantheon of proteins involved in surveilling translational fidelity and controlling gene expression in higher eukaryotes.

A novel de novo variant in CASK causes a severe neurodevelopmental disorder that masks the phenotype of a novel de novo variant in EEF2.

We report a 9-year-old Spanish boy with severe psychomotor developmental delay, short stature, microcephaly and abnormalities of the brain morphology, including cerebellar atrophy. Whole-exome sequencing (WES) uncovered two novel de novo variants, a hemizygous variant in CASK (Calcium/Calmodulin Dependent Serine Protein Kinase) and a heterozygous variant in EEF2 (Eukaryotic Translation Elongation Factor 2). CASK gene encodes the peripheral plasma membrane protein CASK that is a scaffold protein located at the synapses in the brain. The c.2506-6 A > G CASK variant induced two alternative splicing events that account for the 80% of the total transcripts, which are likely to be degraded by NMD. Pathogenic variants in CASK have been associated with severe neurological disorders such as mental retardation with or without nystagmus also called FG syndrome 4 (FGS4), and intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH). Heterozygous variants in EEF2, which encodes the elongation factor 2 (eEF2), have been associated to Spinocerebellar ataxia 26 (SCA26) and more recently to a childhood-onset neurodevelopmental disorder with benign external hydrocephalus. The yeast model system used to investigate the functional consequences of the c.34 A > G EEF2 variant supported its pathogenicity by demonstrating it affects translational fidelity. In conclusion, the phenotype associated with the CASK variant is more severe and masks the milder phenotype of EEF2 variant.

Deep mutational analysis of elongation factor eEF2 residues implicated in human disease to identify functionally important contacts with the ribosome.

An emerging body of research is revealing mutations in elongation factor eEF2 that are implicated in both inherited and de novo neurodevelopmental disorders. Previous structural analysis has revealed that most pathogenic amino acid substitutions map to the three main points of contact between eEF2 and critical large subunit rRNA elements of the ribosome, specifically to contacts with Helix 69, Helix 95, also known as the sarcin-ricin loop, and Helix 43 of the GTPase-associated center. In order to further investigate these eEF2-ribosome interactions, we identified a series of yeast eEF2 amino acid residues based on their proximity to these functionally important rRNA elements. Based on this analysis, we constructed mutant strains to sample the full range of amino acid sidechain biochemical properties, including acidic, basic, nonpolar, and deletion (alanine) residues. These were characterized with regard to their effects on cell growth, sensitivity to ribosome-targeting antibiotics, and translational fidelity. We also biophysically characterized one mutant from each of the three main points of contact with the ribosome using CD. Collectively, our findings from these studies identified functionally critical contacts between eEF2 and the ribosome. The library of eEF2 mutants generated in this study may serve as an important resource for biophysical studies of eEF2/ribosome interactions going forward.

Biomotors, viral assembly, and RNA nanobiotechnology: Current achievements and future directions.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.

The promises and pitfalls of specialized ribosomes.

The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.

EGR1 Upregulation during Encephalitic Viral Infections Contributes to Inflammation and Cell Death.

Early growth response 1 (EGR1) is an immediate early gene and transcription factor previously found to be significantly upregulated in human astrocytoma cells infected with Venezuelan equine encephalitis virus (VEEV). The loss of EGR1 resulted in decreased cell death but had no significant impact on viral replication. Here, we extend these studies to determine the impacts of EGR1 on gene expression following viral infection. Inflammatory genes CXCL3, CXCL8, CXCL10, TNF, and PTGS2 were upregulated in VEEV-infected cells, which was partially dependent on EGR1. Additionally, transcription factors, including EGR1 itself, as well as ATF3, FOS, JUN, KLF4, EGR2, and EGR4 were found to be partially transcriptionally dependent on EGR1. We also examined the role of EGR1 and the changes in gene expression in response to infection with other alphaviruses, including eastern equine encephalitis virus (EEEV), Sindbis virus (SINV), and chikungunya virus (CHIKV), as well as Zika virus (ZIKV) and Rift Valley fever virus (RVFV), members of the Flaviviridae and Phenuiviridae families, respectively. EGR1 was significantly upregulated to varying degrees in EEEV-, CHIKV-, RVFV-, SINV-, and ZIKV-infected astrocytoma cells. Genes that were identified as being partially transcriptionally dependent on EGR1 in infected cells included ATF3 (EEEV, CHIKV, ZIKV), JUN (EEEV), KLF4 (SINV, ZIKV, RVFV), CXCL3 (EEEV, CHIKV, ZIKV), CXCL8 (EEEV, CHIKV, ZIKV, RVFV), CXCL10 (EEEV, RVFV), TNF-α (EEEV, ZIKV, RVFV), and PTGS2 (EEEV, CHIKV, ZIKV). Additionally, inhibition of the inflammatory gene PTGS2 with Celecoxib, a small molecule inhibitor, rescued astrocytoma cells from VEEV-induced cell death but had no impact on viral titers. Collectively, these results suggest that EGR1 induction following viral infection stimulates multiple inflammatory mediators. Managing inflammation and cell death in response to viral infection is of utmost importance, especially during VEEV infection where survivors are at-risk for neurological sequalae.

Identifying Inhibitors of -1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses.

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of -1 PRF in SARS-CoV-2 also inhibited -1 PRF in a range of bat CoVs-the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed -1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.

PERK Is Critical for Alphavirus Nonstructural Protein Translation.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis. Previous work indicated that VEEV infection induced early growth response 1 (EGR1) expression, leading to cell death via the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) arm of the unfolded protein response (UPR) pathway. Loss of PERK prevented EGR1 induction and decreased VEEV-induced death. The results presented within show that loss of PERK in human primary astrocytes dramatically reduced VEEV and eastern equine encephalitis virus (EEEV) infectious titers by 4-5 log10. Loss of PERK also suppressed VEEV replication in primary human pericytes and human umbilical vein endothelial cells, but it had no impact on VEEV replication in transformed U87MG and 293T cells. A significant reduction in VEEV RNA levels was observed as early as 3 h post-infection, but viral entry assays indicated that the loss of PERK minimally impacted VEEV entry. In contrast, the loss of PERK resulted in a dramatic reduction in viral nonstructural protein translation and negative-strand viral RNA production. The loss of PERK also reduced the production of Rift Valley fever virus and Zika virus infectious titers. These data indicate that PERK is an essential factor for the translation of alphavirus nonstructural proteins and impacts multiple RNA viruses, making it an exciting target for antiviral development.

Programmed -1 Ribosomal Frameshifting in coronaviruses: A therapeutic target.

Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.

De Novo variants in EEF2 cause a neurodevelopmental disorder with benign external hydrocephalus.

Eukaryotic translation elongation factor 2 (eEF2) is a key regulatory factor in gene expression that catalyzes the elongation stage of translation. A functionally impaired eEF2, due to a heterozygous missense variant in the EEF2 gene, was previously reported in one family with spinocerebellar ataxia-26 (SCA26), an autosomal dominant adult-onset pure cerebellar ataxia. Clinical exome sequencing identified de novo EEF2 variants in three unrelated children presenting with a neurodevelopmental disorder (NDD). Individuals shared a mild phenotype comprising motor delay and relative macrocephaly associated with ventriculomegaly. Populational data and bioinformatic analysis underscored the pathogenicity of all de novo missense variants. The eEF2 yeast model strains demonstrated that patient-derived variants affect cellular growth, sensitivity to translation inhibitors and translational fidelity. Consequently, we propose that pathogenic variants in the EEF2 gene, so far exclusively associated with late-onset SCA26, can cause a broader spectrum of neurologic disorders, including childhood-onset NDDs and benign external hydrocephalus.

Two Ribosomes Are Better Than One... Sometimes.

In this issue of Molecular Cell, Meydan and Guydosh present an elegant and rigorous addition to the exciting investigation of the roles played by ribosome collisions in eukaryotic translation and cellular homeostasis.

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS coronavirus 2 (SARS-CoV-2).

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS-CoV-2.

17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism.

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

The Expanding Riboverse.

Subverting the conventional concept of "the" ribosome, a wealth of information gleaned from recent studies is revealing a much more diverse and dynamic ribosomal reality than has traditionally been thought possible. A diverse array of researchers is collectively illuminating a universe of heterogeneous and adaptable ribosomes harboring differences in composition and regulatory capacity: These differences enable specialization. The expanding universe of ribosomes not only comprises an incredible richness in ribosomal specialization between species, but also within the same tissues and even cells. In this review, we discuss ribosomal heterogeneity and speculate how the emerging understanding of the ribosomal repertoire is impacting the biological sciences today. Targeting pathogen-specific and pathological "diseased" ribosomes promises to provide new treatment options for patients, and potential applications for "designer ribosomes" are within reach. Our deepening understanding of and ability to manipulate the ribosome are establishing both the technological and theoretical foundations for major advances for the 21st century and beyond.

Translational recoding signals: Expanding the synthetic biology toolbox.

Innovation follows discovery. If the 20th century was a golden age of discovery in the biomolecular biosciences, the current century may be remembered by the explosion of beneficial devices and therapies conceived by the bioengineers of the era. Much as the development of solid-state electronic components made possible the information revolution, the rational combining of millions of basic molecular control modules will enable the development of highly sophisticated biomachines that will make today's smartphones appear rudimentary. The molecular toolbox is already well-stocked, particularly in our ability to manipulate DNA, control transcription, generate functionally novel hybrid proteins, and expand the genetic code to include unnatural amino acids. This review focuses on how RNA-based regulatory modules that direct alternative readings of the genetic code can be employed as basic circuit components to expand our ability to control gene expression.