Start-stop elements are translation regulatory elements in 5 ' untranslated regions (UTR) of eukaryotic transcripts, consisting of a start codon immediately followed by a stop codon. In contrast to canonical upstream Translons (uTranslons), they exclude elongation which creates unique properties. We conducted a comprehensive, carefully controlled comparison of human start-stop elements and uTranslons both at a genome-wide level and with targeted reporter assays. We found that start-stops and uTranslons were similar with respect to their presence in the 5 ' UTRs of hundreds of genes, in particular transcription factors and signaling molecules, the low transcript levels of the corresponding genes, short RNA half-lives, and the negative effect on downstream translation. However, start-stop containing genes were translationally even more repressed than genes with uTranslons. Analysing the start-stop architecture and diverse ribosome footprinting datasets, we found evidence for a start-stop-specific mechanism that involves repeat cycling between initiation, termination, ribosome splitting, and 60S rejoining-a process possibly modulated by ASCC3 and eIF1. This cycling explained increased ribosome retention at start-stops and was-in contrast to ribosome retention at uTranslons-independent of the global initiation state. Finally, we showed that the start-stop element in human ATF4 augments the core regulatory model by controlling translation of the uTranslons.

C9ORF72 is involved in multiple neuronal functions and a major factor in Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disease. Pathogenicity is implemented through an intronic repeat expansion in the gene’s mRNA leader that produces long, repetitive RNA and dipeptides when translated through a non-canonical mechanism. In contrast, despite the presence of ribosome-occupied, regulatory elements in the mRNA leader, nothing is known about C9ORF72 translation regulation under normal conditions. Surprisingly, when analyzing a series of mutants of the C9ORF72 mRNA leader, we found that translation of C9ORF72 is tightly regulated through a multi-layer system. First, non-canonical, multi-initiation upstream open reading frames (nc-uORFs) in all three frames, a start-stop element in frame 2, and a canonical uORF in frame 1work together to keep baseline C9ORF72 translation very low. Second, a strong secondary structure enhances these repressive elements, mainly the two nc-uORFs in frames 0 and 2. Finally, as initiation at the nc-uORFs reduces initiation at the downstream start-stop, the nc-uORFs effectively dampen the start-stop’s repressive function, forming a feedforward loop. We hypothesize that this buffered repressor system has likely evolved to ensure reliable, noise-insensitive expression of this critical regulator of neuronal function. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R35GM127089, T32AG052909

An increased level of phosphorylation of eukaryotic translation initiation factor 2 subunit-α (eIF2α, encoded by EIF2S1; eIF2α-p) coupled with decreased guanine nucleotide exchange activity of eIF2B is a hallmark of the ‘canonical’ integrated stress response (c-ISR)1. It is unclear whether impaired eIF2B activity in human diseases including leukodystrophies2, which occurs in the absence of eIF2α-p induction, is synonymous with the c-ISR. Here we describe a mechanism triggered by decreased eIF2B activity, distinct from the c-ISR, which we term the split ISR (s-ISR). The s-ISR is characterized by translational and transcriptional programs that are different from those observed in the c-ISR. Opposite to the c-ISR, the s-ISR requires eIF4E-dependent translation of the upstream open reading frame 1 and subsequent stabilization of ATF4 mRNA. This is followed by altered expression of a subset of metabolic genes (for example, PCK2), resulting in metabolic rewiring required to maintain cellular bioenergetics when eIF2B activity is attenuated. Overall, these data demonstrate a plasticity of the mammalian ISR, whereby the loss of eIF2B activity in the absence of eIF2α-p induction activates the eIF4E–ATF4–PCK2 axis to maintain energy homeostasis. A study describes the split integrated stress response, a cellular stress response mechanism characterized by reduced eIF2B activity without eIF2α phosphorylation, which activates the eIF4E–ATF4–PCK2 axis, enabling metabolic reprogramming.

Amyotrophic lateral sclerosis damages proteostasis, affecting spinal and upper motor neurons earlier than a subset of cranial motor neurons. To aid disease understanding, we exposed induced cranial and spinal motor neurons (iCrMNs and iSpMNs) to proteotoxic stress, under which iCrMNs showed superior survival, quantifying the transcriptome and proteome for >8,200 genes at 0, 12, and 36 h. Two-thirds of the proteome showed cell-type differences. iSpMN-enriched proteins related to DNA/RNA metabolism, and iCrMN-enriched proteins acted in the endoplasmic reticulum (ER)/ER chaperone complex, tRNA aminoacylation, mitochondria, and the plasma/synaptic membrane, suggesting that iCrMNs expressed higher levels of proteins supporting proteostasis and neuronal function. When investigating the increased proteasome levels in iCrMNs, we showed that the activity of the 26S proteasome, but not of the 20S proteasome, was higher in iCrMNs than in iSpMNs, even after a stress-induced decrease. We identified Ublcp1 as an iCrMN-specific regulator of the nuclear 26S activity.

Understanding the molecular mechanisms underpinning diverse vaccination responses is critical for developing efficient vaccines. Molecular subtyping can offer insights into heterogeneous nature of responses and aid in vaccine design. We analyzed multi-omic data from 62 haemagglutinin seasonal influenza vaccine recipients (2019-2020), including transcriptomics, proteomics, glycomics, and metabolomics data collected pre-vaccination. We performed a subtyping analysis on the integrated data revealing five subtypes with distinct molecular signatures. These subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining the effectiveness of seasonal vaccines. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

The COVID-19 pandemic, triggered by severe acute respiratory syndrome coronavirus 2, has affected millions of people worldwide. Much research has been dedicated to our understanding of COVID-19 disease heterogeneity and severity, but less is known about recovery associated changes. To address this gap in knowledge, we quantified the proteome from serum samples from 29 COVID-19 convalescents and 29 age-, race-, and sex-matched healthy controls. Samples were acquired within the first months of the pandemic. Many proteins from pathways known to change during acute COVID-19 illness, such as from the complement cascade, coagulation system, inflammation and adaptive immune system, had returned to levels seen in healthy controls. In comparison, we identified 22 and 15 proteins with significantly elevated and lowered levels, respectively, amongst COVID-19 convalescents compared to healthy controls. Some of the changes were similar to those observed for the acute phase of the disease, i.e. elevated levels of proteins from hemolysis, the adaptive immune systems, and inflammation. In contrast, some alterations opposed those in the acute phase, e.g. elevated levels of CETP and APOA1 which function in lipid/cholesterol metabolism, and decreased levels of proteins from the complement cascade (e.g. C1R, C1S, and VWF), the coagulation system (e.g. THBS1 and VWF), and the regulation of the actin cytoskeleton (e.g. PFN1 and CFL1) amongst COVID-19 convalescents. We speculate that some of these shifts might originate from a transient decrease in platelet counts upon recovery from the disease. Finally, we observed race-specific changes, e.g. with respect to immunoglobulins and proteins related to cholesterol metabolism.

Understanding the molecular mechanisms that underpin diverse vaccination responses is a critical step toward developing efficient vaccines. Molecular subtyping approaches can offer valuable insights into the heterogeneous nature of responses and aid in the design of more effective vaccines. In order to explore the molecular signatures associated with the vaccine response, we analyzed baseline transcriptomics data from paired samples of whole blood, proteomics and glycomics data from serum, and metabolomics data from urine, obtained from influenza vaccine recipients (2019-2020 season) prior to vaccination. After integrating the data using a network-based model, we performed a subtyping analysis. The integration of multiple data modalities from 62 samples resulted in five baseline molecular subtypes with distinct molecular signatures. These baseline subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation across subtypes in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these significant differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining vaccine response and efficacy. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4’s induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4’s translational control is more complex than originally described, underpinning its key role in diverse biological processes.

Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines. Key features • Protocol for fractionation of active nuclear and cytoplasmic proteasome complexes. • Native electrophoresis and fluorescence-based in-gel activity assay, which allows the visualization and quantification of active complexes within the acrylamide gel matrix. • In-gel protein denaturation followed by transfer of complexes to PVDF membrane, which allows the analysis of complexes' abundance using antibodies.

During embryonic development, cell-fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors of both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study cardiopharyngeal fate specification with cellular resolution, as only two bilateral pairs of multipotent cardiopharyngeal progenitors give rise to the heart and to the pharyngeal muscles (also known as atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented and asymmetric divisions. Here, we identify the primed gene ring finger 149 related (Rnf149-r), which later becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss of Rnf149-r function impairs atrial siphon muscle morphogenesis, and downregulates Tbx1/10 and Ebf, two key determinants of pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of the loss of FGF/MAPK signaling in the cardiopharyngeal lineage, and an integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations has identified a significant overlap between candidate FGF/MAPK and Rnf149-r target genes. However, functional interaction assays suggest that Rnf149-r does not directly modulate the activity of the FGF/MAPK/Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF/MAPK signaling on shared targets, as well as on FGF/MAPK-independent targets through (a) separate pathway(s).

Zika virus (ZIKV) and dengue virus (DENV) are two closely related flaviviruses with similar symptoms. However, due to the implications of ZIKV infections for pregnancy outcomes, understanding differences in their molecular impact on the host is of high interest. Viral infections change the host proteome, including post-translational modifications. As modifications are diverse and of low abundance, they typically require additional sample processing which is not feasible for large cohort studies. Therefore, we tested the potential of next-generation proteomics data in its ability to prioritize specific modifications for later analysis. We re-mined published mass spectra from 122 serum samples from ZIKV and DENV patients for the presence of phosphorylated, methylated, oxidized, glycosylated/glycated, sulfated, and carboxylated peptides. We identified 246 modified peptides with significantly differential abundance in ZIKV and DENV patients. Amongst these, methionine-oxidized peptides from apolipoproteins and glycosylated peptides from immunoglobulin proteins were more abundant in ZIKV patient serum and generate hypotheses on the potential roles of the modification in the infection. The results demonstrate how data-independent acquisition techniques can help prioritize future analyses of peptide modifications.

Various mechanisms controlling ribosome scanning and initiation have been identified within the 5’ untranslated region (UTR) of eukaryotic genes, including upstream open reading frames, specific sequence motifs, and secondary structures. We describe the impact and distinct characteristics of a translation regulatory element consisting of a start codon immediately followed by a stop codon. While to date this start-stop element has been described only for specific cases, we identified them in the 5’UTR of hundreds of human genes whereby their general impact on translation control in mammalian cells has been unnoticed. We show that the element is evolutionarily conserved and represses translation of the downstream open reading frames. While having no apparent impact on translation termination or RNA stability, start-stops efficiently halt ribosomes and act as a barrier for the scanning of the small ribosomal subunit. Start-stop element occurs in many transcription factors and key signaling genes, and we highlight routes in which they can affect cellular fate. We investigate the start-stop element in several genes, i.e. MORF4L1 , IFNAR1 , SLC39A1 , and PSPC1 , and delineate the role of the start-stop in translation regulation of ATF4 mRNA. Thus, start-stops uniquely modulate translation through elongation-less ribosome stalling.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset neurodegenerative disease without cure or understanding of its molecular basis. It is characterized by progressive loss of spinal and upper motor neurons, but surprising resistance of cranial motor neurons until the late stages of the disease. There is no known single-gene cause of ALS, suggesting a failure of the entire cellular system to maintain proteostasis. To generate new hypotheses on the molecular differences between cranial and spinal motor neurons that underlie ALS-sensitivity, we exploited a unique platform that generates highly pure populations of induced cranial and spinal motor neurons (iCrMN and iSpMN). Exposing both iCrMNs and iSpMNs to proteotoxic stress, we monitored transcriptome and proteome changes over 36 hours and identified significant hundreds of statistically significant differences at either or both the RNA and protein level from a core set of 8,206 genes with complete data. While we observed a response to misfolding stress coordinated between transcript and protein changes, differences between the cell type were specific to RNA and protein expression. iCrMNs and iSpMNs differed in expression of many membrane proteins, including synaptic proteins, solute carriers, channels, and signaling molecules, as well as genes involved in ribosome biogenesis and subunits of the core proteasome. Differences exclusive to the transcriptome involved chaperones and the tricarboxylic acid cycle. Further, we found that the superior proteostasis capability of iCrMNs might, in part, be caused due to higher activity and abundance of the fully assembled 26S/30S proteasome in the nucleus, pointing to an increased role of ubiquitin-mediated, targeted protein degradation. The formation of the 26S/30S proteasome was highly dynamic under stress, likely mediated by a surplus of regulatory particles that attached and detached based on the action of proteasome regulators. Several lines of evidence supported this notion and described an intricate network of differentially expressed proteasome regulators and ubiquitination enzymes that defined the iCrMNand iSpMN-specific ability to maintain proteostasis.

AbstractDuring embryonic development, cell fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors to both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study the cardiopharyngeal fate specification with cellular resolution, as only 2 pairs of cardiopharyngeal multipotent progenitors give rise to the heart and to pharyngeal muscles (aka atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented asymmetric divisions. Here, we identify the primed gene Rnf149-related (Rnf149-r), which becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss knock-out of Rnf149-r function impairs atrial siphon muscle morphogenesis, and down-regulates Tbx1/10 and Ebf, two key determinants of the pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of loss of FGF-MAPK signaling in the cardiopharyngeal lineage, and integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations identified a significant overlap between FGF-MAPK and Rnf149-r targets. However, functional interaction assays suggested the Rnf149-r does not directly modulate the activity of the FGF-MAPK-Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF-MAPK signaling on shared targets, as well as on FGF-MAPK-independent targets through (a) separate pathway(s).

The serological response to the influenza virus vaccine is highly heterogeneous for reasons that are not entirely clear. While the impact of demographic factors such as age, body mass index (BMI), sex, prior vaccination and titer levels are known to impact seroconversion, they only explain a fraction of the response. To identify signatures of the vaccine response, we analyzed 273 protein levels from 138 serum samples of influenza vaccine recipients (2019–2020 season). We found that levels of proteins functioning in cholesterol transport were positively associated with seroconversion, likely linking to the known impact of BMI. When adjusting seroconversion for the demographic factors, we identified additional, unexpected signatures: proteins regulating actin cytoskeleton dynamics were significantly elevated in participants with high adjusted seroconversion. Viral strain specific analysis showed that this trend was largely driven by the H3N2 strain. Further, we identified complex associations between adjusted seroconversion and other factors: levels of proteins of the complement system associated positively with adjusted seroconversion in younger participants, while they were associated negatively in the older population. We observed the opposite trends for proteins of high density lipoprotein remodeling, transcription, and hemostasis. In sum, careful integrative modeling can extract new signatures of seroconversion from highly variable data that suggest links between the humoral response as well as immune cell communication and migration.

ABSTRACTResponse to vaccination can vary significantly from person to person. A key to improving vaccine design and vaccination strategy is to understand the mechanism behind this variation. The role of glycosylation, a critical modulator of immunity, is unknown in determining vaccine responses. To gain insight into the association between glycosylation and vaccine-induced antibody levels we profiled the pre- and post-vaccination serum protein glycomes of 160 Caucasian adults receiving the FLUZONE™ influenza vaccine during the 2019-2020 influenza season. Using lectin microarrays, we observed that pre-vaccination levels of Lewis A antigen (Lea) are significantly higher in people who did not mount significant antibody responses, when compared to responders. Glycoproteomic analysis showed that Lea-bearing proteins are enriched in complement activation pathways, suggesting a potential role of glycosylation in tuning the activities of complement proteins, which may be implicated in mounting vaccine responses. We also observed post-vaccination increases in sialyl Lewis X antigen (sLex) and decreases in high mannose glycans among high responders, which were not observed in non-responders. This data suggests that the immune system may actively modulate glycosylation as part of its effort to establish effective protection post-vaccination.

A key to improving vaccine design and vaccination strategy is to understand the mechanism behind the variation of vaccine response with host factors. Glycosylation, a critical modulator of immunity, has no clear role in determining vaccine responses. To gain insight into the association between glycosylation and vaccine-induced antibody levels, we profiled the pre- and postvaccination serum protein glycomes of 160 Caucasian adults receiving the FLUZONE influenza vaccine during the 2019-2020 influenza season using lectin microarray technology. We found that prevaccination levels of Lewis A antigen (Lea) are significantly higher in nonresponders than responders. Glycoproteomic analysis showed that Lea-bearing proteins are enriched in complement activation pathways, suggesting a potential role of glycosylation in tuning the activities of complement proteins, which may be implicated in mounting vaccine responses. In addition, we observed a postvaccination increase in sialyl Lewis X antigen (sLex) and a decrease in high mannose glycans among high responders, which were not observed in nonresponders. These data suggest that the immune system may actively modulate glycosylation as part of its effort to establish effective protection postvaccination.

Host-associated microbiomes harbour hundreds of bacterial species that co-occur, creating the opportunity for manifold bacteria–bacteria interactions, which in turn contribute to the overall community structure. The mechanisms that underlie this self-organization among bacteria remain largely elusive. Here, we studied bacterial interactions in the phyllosphere microbiota. We screened for microbial interactions in planta by adding 200 endogenous strains individually to a 15-member synthetic community and tracking changes in community composition upon colonization of the model plant Arabidopsis. Ninety percent of the identified interactions in planta were negative, and phylogenetically closely related strains elicited consistent effects on the synthetic community, providing support for trait conservation. Community changes could be largely explained by binary interactions; however, we also identified a higher-order interaction of more than two interacting strains. We further focused on a prominent interaction between two members of the Actinobacteria. In the presence of Aeromicrobium Leaf245, the population of Nocardioides Leaf374 was reduced by almost two orders of magnitude. We identified a potent antimicrobial peptidase in Aeromicrobium Leaf245, which resulted in Nocardioides Leaf374 lysis. A respective Leaf245 mutant strain was necessary and sufficient to restore Nocardioides colonization in planta, demonstrating that direct bacteria–bacteria interactions were responsible for the population shift originally observed. Our study highlights the power of synthetic community screening and uncovers a strong microbial interaction that occurs despite a spatially heterogeneous environment. Bacteria–bacteria interactions and resultant microbiota changes were characterized using a synthetic phyllosphere community in planta.

Cellular stressors are abundant and diverse, and they can be acute or chronic and mild or intense. Accordingly, the stress response is complex, involving transcription, RNA stability, translation, protein turnover, and changes in interactions, as well as modifications and re-localization of molecules. Different response types have their specific triggers, regulators, and dynamics, with the common goal of reestablishing cellular health.