Dynamic Regulation of the Nexus Between Stress Granules, Roquin, and Regnase-1 Underlies the Molecular Pathogenesis of Warfare Vesicants

The use of chemical warfare agents is prohibited but they have been used in recent Middle Eastern conflicts. Their accidental exposure (e.g. arsenical lewisite) is also known and causes extensive painful cutaneous injury. However, their molecular pathogenesis is not understood. Here, we demonstrate that a nexus of stress granules (SGs), integrated stress, and RNA binding proteins (RBPs) Roquin and Reganse-1 play a key role. Lewisite and its prototype phenylarsine oxide (PAO) induce SG assembly in skin keratinocytes soon after exposure, which associate with various RBPs and translation-related proteins. SG disassembly was detected several hours after exposure. The dynamics of SG assembly-disassembly associates with the chemical insult and cell damage. Enhanced Roquin and Regnase-1 expression occurs when Roquin was recruited to SGs and Regnase-1 to the ribosome while in the disassembling SGs their expression is decreased with consequent induction of inflammatory mediators. SG-targeted protein translational control is regulated by the phosphorylation-dependent activation of eukaryotic initiation factors 2α (eIF2α). Treatment with integrated stress response inhibitor (ISRIB), which blocks eIF2α phosphorylation, impacted SG assembly dynamics. Topical application of ISRIB attenuated the inflammation and tissue disruption in PAO-challenged mice. Thus, the dynamic regulation of these pathways provides underpinning to cutaneous injury and identify translational therapeutic approach for these and similar debilitating chemicals.

Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

eIF4A2 targets developmental potency and histone H3.3 transcripts for translational control of stem cell pluripotency

Translational control has emerged as a fundamental regulatory layer of proteome complexity that governs cellular identity and functions. As initiation is the rate-limiting step of translation, we carried out an RNAi screen for key translation initiation factors required to maintain embryonic stem cell (ESC) identity. We identified eIF4A2 and defined its mechanistic action through Rps26-independent and -dependent ribosomes in translation initiation activation of mRNAs encoding pluripotency factors and the histone variant H3.3 with demonstrated roles in maintaining stem cell pluripotency. eIF4A2 also mediates translation initiation activation of Ddx6, which acts together with eIF4A2 to restrict the totipotent 2-cell transcription program in ESCs through Zscan4 mRNA degradation and translation repression. Accordingly, knockdown of eIF4A2 disrupts ESC proteome causing the loss of ESC identity. Collectively, we establish a translational paradigm of the protein synthesis of pluripotency transcription factors and epigenetic regulators imposed on their established roles in controlling pluripotency.

The Role of the Universally Conserved ATPase YchF/Ola1 in Translation Regulation during Cellular Stress

The ability to respond to metabolic or environmental changes is an essential feature in all cells and involves both transcriptional and translational regulators that adjust the metabolic activity to fluctuating conditions. While transcriptional regulation has been studied in detail, the important role of the ribosome as an additional player in regulating gene expression is only beginning to emerge. Ribosome-interacting proteins are central to this translational regulation and include universally conserved ribosome interacting proteins, such as the ATPase YchF (Ola1 in eukaryotes). In both eukaryotes and bacteria, the cellular concentrations of YchF/Ola1 determine the ability to cope with different stress conditions and are linked to several pathologies in humans. The available data indicate that YchF/Ola1 regulates the stress response via controlling non-canonical translation initiation and via protein degradation. Although the molecular mechanisms appear to be different between bacteria and eukaryotes, increased non-canonical translation initiation is a common consequence of YchF/Ola1 regulated translational control in E. coli and H. sapiens. In this review, we summarize recent insights into the role of the universally conserved ATPase YchF/Ola1 in adapting translation to unfavourable conditions.

Translational control in cell ageing: an update

Cellular ageing is one of the main drivers of organismal ageing and holds keys towards improving the longevity and quality of the extended life. Elucidating mechanisms underlying the emergence of the aged cells as well as their altered responses to the environment will help understanding the evolutionarily defined longevity preferences across species with different strategies of survival. Much is understood about the role of alterations in the DNA, including many epigenetic modifications such as methylation, in relation to the aged cell phenotype. While transcriptomes of the aged cells are beginning to be better-characterised, their translational responses remain under active investigation. Many of the translationally controlled homeostatic pathways are centred around mitigation of DNA damage, cell stress response and regulation of the proliferative potential of the cells, and thus are critical for the aged cell function. Translation profiling-type studies have boosted the opportunities in discovering the function of protein biosynthesis control and are starting to be applied to the aged cells. Here, we provide a summary of the current knowledge about translational mechanisms considered to be commonly altered in the aged cells, including the integrated stress response-, mechanistic target of Rapamycin- and elongation factor 2 kinase-mediated pathways. We enlist and discuss findings of the recent works that use broad profiling-type approaches to investigate the age-related translational pathways. We outline the limitations of the methods and the remaining unknowns in the established ageing-associated translation mechanisms, and flag translational mechanisms with high prospective importance in ageing, for future studies.

RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

Background and Aims: Translational regulation involve the coordinated actions of RNA binding proteins (RBPs) and non-coding RNAs. For efficient translation, the mRNA needs to be circularized. While RNA binding proteins and translation factors have been shown to regulate the circularization, the role of lncRNAs in the process is not yet defined. Methods: We first performed RNA-seq and RNA-immunoprecipitation coupled-Seq (RIP-Seq) to identify differentially expressed lncRNA and mRNA in RBM3 overexpressing cell lines. We manipulated lncRNA expression in the cells and determined effects on gene expression and cell viability and motility. The studies were confirmed in vivo in intestine specific RBM3 transgenic and RBM3 knockout mouse models. Results: In comparing the RNA-Seq and RIP-Seq datasets, we identified increased expression of lncRNA LSAMP-3 and Flii-1 that bind to RBM3. In addition, there was an increase in expression of epithelial mesenchymal transition and angiogenesis markers following RBM3 overexpression. Moreover, modeling studies suggest that these lncRNAs formed kissing-loop interactions on target mRNAs including transcripts that encode epithelial mesenchymal transition and angiogenesis. While RBM3 transgenic mice showed increased LSAMP-3 and Flii-1, this was reduced in the RBM3 knockout mice. Also, RBM3 overexpression increased tumor xenograft growth, which was suppressed by knockdown of the lncRNAs. Also, knockdown of endogenous RBM3 specifically in the intestine suppressed azoxymethane-dextran sodium sulfate driven colitis-associated cancers, with a corresponding reduction in the expression of lncRNAs and transcripts that encode epithelial mesenchymal transition and angiogenesis. Conclusion: We propose that RBPs such as RBM3 mediate their function through regulatory lncRNAs that enable circularization to control translation.

Genome instability drives epistatic adaptation in the human pathogen Leishmania

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2′-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

AZFa Y Gene, DDX3Y, Evolved Novel Testis Transcript Variants in Primates with Proximal 3´UTR Polyadenylation for Germ Cell Specific Translation

Translational control is a major level of gene expression regulation in the male germ line. DDX3Y located in the AZFa region of the human Y chromosome encodes a conserved RNA helicase important for translational control at the G1-S phase of the cell cycle. In human, DDX3Y protein is expressed only in premeiotic male germ cells. In primates, DDX3Y evolved a second promoter producing novel testis-specific transcripts. Here, we show primate species-specific use of alternative polyadenylation (APA) sites for the testis-specific DDX3Y transcript variants. They have evolved first in the 3´UTRs of primate DDX3Y transcripts. A distal APA site is used for polyadenylation of DDX3Y testis transcripts in Callithrix jacchus; two proximal APAs in Macaca mulatta, in Pan trogloydates and in human. This shift corresponds with a significant increase of DDX3Y protein expression in the macaque testis and kidney tissue. In chimpanzee and human, shift to predominant use of the most proximal APA site is associated with translation of these DDX3Y transcripts in only premeiotic male germ cells. We therefore assume evolution of a positive selection process for functional DDX3Y testis transcripts in these primates to promote increase of their stability and balancing translation efficiency especially in the male germ line.

Disorder at the Start: The Contribution of Dysregulated Translation Initiation to Cancer Therapy Resistance

Translation of cellular RNA to protein is an energy-intensive process through which synthesized proteins dictate cellular processes and function. Translation is regulated in response to extracellular effectors and availability of amino acids intracellularly. Most eukaryotic mRNA rely on the methyl 7-guanosine (m7G) nucleotide cap to recruit the translation machinery, and the uncoupling of translational control that occurs in tumorigenesis plays a significant role in cancer treatment response. This article provides an overview of the mammalian translation initiation process and the primary mechanisms by which it is regulated. An outline of how deregulation of initiation supports tumorigenesis and how initiation at a downstream open reading frame (ORF) of Tousled-like kinase 1 (TLK1) leads to treatment resistance is discussed.

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.