Downregulation Of Translation Research Articles

Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1.

Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process. By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production. The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Abstract 5849: Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy

Abstract Repurposed drugs are undergoing a renaissance in the era of genomics, particularly in oncology. Over 2,300 FDA-approved and natural product small molecules now exist, but challenges remain in identifying which cancer patients may best benefit from such drugs. To best position repurposed drugs, mechanisms of action and resistance to the drug in a cancer cell setting must be determined. Hydroxychloroquine (HCQ) and chloroquine are repurposed drugs reported to disrupt autophagy, a molecular recycling pathway which is essential for tumor cell survival, chemotherapeutic resistance, and stem cell phenotypes. However, oncology trials with chloroquine have no biomarker or other genetic test to categorize patients who may best respond to HCQ therapy. It is unclear how tumors mechanistically evolve resistance to HCQ. We pursued a multi-omic strategy in OVCAR3 ovarian cancer and CCL218 colorectal cancer cells. Two genome-scale screens were performed. In the forward genetic screen, cell populations were passaged in 93-independent cultures for 15 drug pulse-chases with HCQ or vehicle control. Evolved cells were collected and processed for bulk RNA-seq, exome-seq, and single cell RNA-seq (scRNA-seq). In the reverse genetic screen, a pooled Brunello CRISPR-Cas9 library of 19,114 genes targeted by multiple sgRNAs was used in cells over three pulse-chases of HCQ or vehicle control treatments. HCQ evolved cells displayed remarkably few mutational differences, but substantial transcriptional differences. Bulk transcriptomes revealed multiple broad-spectrum pathways associated with resistance to HCQ, including upregulation of glycolysis, exocytosis, and chromosome condensation/segregation, as well as downregulation of translation and apoptosis. Analysis of scRNA-seq data indicated cell clusters were differentially reliant on cytoskeletal regulation, DNA repair, and metabolism. The genome-scale Cas9 screen identified remarkably few autophagy genes. However, an integrated analysis of Cas9 hits with RNA-seq upregulated genes confirmed glycolytic genes KDM3A, ENO2, and PDK3 were essential for HCQ resistance. Additional integrated hits were NEK2, involved in exocytosis, and chromosome condensation/segregation genes CDK1 and CENPA. Transcriptional plasticity was the primary mechanism by which cells evolved resistance to HCQ. Contrary to the widely presumed autophagy-related mode of cell death, the multi-omic analysis instead prioritized glycolytic metabolism, exocytosis, and chromosome condensation/segregation as most involved in cancer cell survival with HCQ. Integrated multi-omic analysis defined high-value biomarkers of HCQ sensitivity and resistance. Our analysis may serve as a model for how to better define the effects and positioning of repurposed drugs in oncology. Citation Format: Madison Clark, Silvia G. Vaena, Joe R. Delaney. Integrated multi-omics of hydroxychloroquine evolved cancer cells reveal survival requires transcriptional upregulation of glycolysis, exocytosis, and chromosome fidelity, but not autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5849.

Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells

Abstract Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations. Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle. Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors. Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions. Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7080.

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells’ transcriptome and proteome profiles.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic β cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during β cell glucose toxicity that impairs expression of proteins with critical roles in β cell function.

Genotype-Phenotype Mapping Via Single-Cell Multi-Omics Identifies Therapeutic Vulnerabilities in Vexas Syndrome

VEXAS (Vacuoles E1 enzyme X-linked Autoinflammatory Somatic) syndrome originates from hematopoietic stem cells (HSCs) carrying a somatic UBA1 p.M41 mutation. UBA1 p.M41 mutation leads to systemic inflammation and bone marrow failure, with poor survival. Other than allogenic stem cell transplantation, no curative therapy is available for VEXAS. This arises from the challenge of defining the phenotypic effects of the UBA1 p.M41 mutation in primary human samples, confounded due to the mixture of wildtype and mutated cells. Thus, it is essential to resolve the admixture of wildtype and UBA1 p.M41 mutated cells to understand the effect of the UBA1 p.M41 mutation in the disease-initiating HSCs. By doing so, we can identify potential mutant-specific therapeutic vulnerabilities to specifically eliminate UBA1 p.M41 HSCs. To map genotype-phenotype relationship in human VEXAS samples, we deployed and developed single-cell multi-omics including Genotyping of Transcriptomes (GoT) and Genotyping of Targeted loci with Chromatin Accessibility (GoT-ChA), allowing the capture of UBA1 genotype, cell identity, transcriptome, chromatin accessibility and signaling pathways at single cell resolution ( Fig.1a). We applied GoT to CD34+ enriched bone marrow mononuclear cells (BMMNCs) from VEXAS primary human samples ( n = 31,406 cells across 6 patients). Integrating UBA1 genotyping with gene expression data showed an enrichment of the mutation in myeloid-committed cells and depletion in B and T cells. Interestingly, this mutation is also enriched in natural killer (NK) cells and plasmacytoid dendritic cells (both originating from lymphoid progenitors), which have not been previously reported. This suggests that VEXAS HSPCs display a skewed differentiation towards myeloid, NK, and plasmacytoid dendritic cells, which may contribute to inflammation in VEXAS. High-resolution mapping of UBA1 p.M41 vs. UBA1 wt HSCs revealed myeloid priming of mutated HSCs. UBA1 p.M41 HSCs showed an increased inflammation signature (Interferon alpha and gamma response pathways) along with increased unfolded protein response (UPR), glycolysis and decreased autophagy pathways. We validated UPR pathway activity by showing the expected increase in XBP1 splicing in UBA1 p.M41 HSPCs using targeted capture of the unconventional splice site. Interestingly, transcriptome data also showed a global translational downregulation accompanied by specific ATF4 target gene enrichment ( Fig.1b). These results suggest a preferential induction of the PERK arm of the UPR. Surface protein expression analysis supported the transcriptomic data, showing upregulation of CD38, CD13 and HLA-DR, denoting myeloid bias and increased inflammatory response in UBA1 p.M41 HSCs. Next, we performed GoT-ChA on CD34 + cells from VEXAS patients (n = 27,894 cells across 3 patients) to study mutant-specific chromatin accessibility. Complementary to the transcriptome data, UBA1 p.M41 HSCs showed increased motif accessibility for TFs associated with myelopoiesis, indicative of myeloid priming. We also observed enrichment for ATF4 and CEPBD TF motifs - downstream of PERK pathway of UPR ( Fig.1b). To validate these findings, we developed GoT-ChA integrated with intracellular protein capture and demonstrated an increased expression of phosphorylated eIF2 and ATF4 proteins in the UBA1 p.M41 HSCs. Finally, to functionally validate the specific role of the PERK pathway on the fitness of UBA1 p.M41 HSCs, we performed assays using mobilized CD34 + cells in-vitro. UBA1 Inhibition in normal CD34 + cells led to ATF4 up-regulation, which correlated with cell survival. The addition of PERK inhibition reduced ATF4 expression and resulted in markedly reduced CD34+ viability. In contrast, inhibiting IRE1 pathway along with UBA1 inhibition did not affect HSC viability. Altogether, we demonstrated that human VEXAS HSCs show a cell-intrinsic inflammatory phenotype and are myeloid biased. Moreover, mutated HSCs tolerate proteome toxicity through activation of the PERK pathway as a part of UPR, providing a fitness advantage to the UBA1 p.M41 mutated HSCs. Targeting the PERK pathway may thus represent a new strategy for overcoming VEXAS syndrome via precision targeting of UBA1 p.M41 mutated HSCs.

Rational design of microRNA-responsive switch for programmable translational control in mammalian cells

Artificial RNA translation modulation usually relies on multiple components, such as RNA binding proteins (RBPs) or microRNAs (miRNAs) for off-switches and double-inverter cascades for on-switches. Recently, translational circular RNAs (circRNAs) were developed as promising alternatives for linear messenger RNAs (mRNAs). However, circRNAs still lack straightforward and programmable translation control strategies. Here, we rationally design a programmable miRNA-responsive internal ribosome entry site (IRES) translation activation and repression (PROMITAR) platform capable of implementing miRNA-based translation upregulation and downregulation in a single RNA construct. Based on the PROMITAR platform, we construct logic gates and cell-type classifier circRNAs and successfully identify desired mammalian cell types. We also demonstrate the potential therapeutic application of our platform for targeted cancer cell killing by encoding a cytotoxic protein in our engineered circRNAs. We expect our platform to expand the toolbox for RNA synthetic biology and provide an approach for potential biomedical applications in the future.

1-Nitropyrene disrupts testicular steroidogenesis via oxidative stress-evoked PERK-eIF2α pathway

Our previous study showed 1-Nitropyrene (1-NP) exposure disrupted testicular testosterone synthesis in mouse, but the exact mechanism needs further investigation. The present research found 4-phenylbutyric acid (4-PBA), an endoplasmic reticulum (ER) stress inhibitor, recovered 1-NP-induced ER stress and testosterone synthases reduction in TM3 cells. GSK2606414, a protein kinase-like ER kinase (PERK) kinase inhibitor, attenuated 1-NP-induced PERK-eukaryotic translation initiation factor 2α (eIF2α) signaling activation and downregulation of steroidogenic proteins in TM3 cells. Both 4-PBA and GSK2606414 attenuated 1-NP-induced steroidogenesis disruption in TM3 cells. Further studies used N-Acetyl-L-cysteine (NAC) as a classical antioxidant to explore whether oxidative stress-activated ER stress mediated 1-NP-induced testosterone synthases reduction and steroidogenesis disruption in TM3 cells and mouse testes. The results showed NAC pretreatment mitigated oxidative stress, and subsequently attenuated ER stress, particularly PERK-eIF2α signaling activation, and downregulation of testosterone synthases in 1-NP-treated TM3 cells. More importantly, NAC extenuated 1-NP-induced testosterone synthesis in vitro and in vivo. The current work indicated that oxidative stress-caused ER stress, particularly PERK-eIF2α pathway activation, mediates 1-NP-downregulated steroidogenic proteins and steroidogenesis disruption in TM3 cells and mouse testes. Significantly, the current study provides a theoretical basis and demonstrates the experimental evidence for the potential application of antioxidant, such as NAC, in public health prevention, particularly in 1-NP-induced endocrine disorder.

Indoxyl sulfate influences susceptibility of proximal tubule epithelial cells to gentamicin cytotoxicity

Background: Patients with chronic kidney disease (CKD) are at increased risk of acute kidney injury (AKI) following exposure to nephrotoxic medications such as gentamicin, which can accelerate the progression of CKD. Therefore, there is a critical need to understand the molecular pathogenesis of acute-on-chronic kidney disease. A hallmark of CKD is the systemic bioaccumulation of uremic solutes, such as the tryptophan metabolite indoxyl sulfate (IS). Accumulating evidence suggests that IS has direct effects on the kidneys that contribute to CKD progression, and the objective of this study was to the determine the effect of IS on proximal tubule epithelial cell (PTEC) and test the hypothesis that IS exposure increases susceptibility to gentamicin-induced cytotoxicity. Methods: Cultured immortalized PTECs (HK-2 cells) were treated with IS and gentamicin at varying concentrations, and cell viability was assessed using the Trypan blue and MTT assays. TMT proteomic profiling with IBAQ quantitation and gene set enrichment analysis was used to identify differentially abundant proteins and cellular processes and pathways that are dysregulated by IS exposure and could mediate the cytotoxic effects of IS and influence susceptibility to gentamicin cytotoxicity. Results: Treatment with 100 μM IS had a modest cytotoxic effect (15-20% cell death after 24 hours over multiple experiments). Proteomic profiling of HK-2 cells identified 10,030 proteins, and IS treatment induced changes in the abundance of 60 proteins, including increased expression of kidney injury molecule-1, an established biomarker of PTEC injury, and VCAM-1, a marker of maladaptive repair after AKI. In contrast, expression of mitochondrial complex V ATPase F o subunits C1, C2, and C3 and mitochondrial ribosomal proteins L18 and L50 were down regulated, and GSEA found that there was an overall decrease in mitochondrial translation following IS exposure. These results suggest that IS is directly cytotoxic to PTECs and negatively impacts mitochondrial dynamics and cellular respiration. Pretreatment with IS increased susceptibility of HK-2 cells to 10 mM gentamicin cytotoxicity as assessed by Trypan blue staining (30.1% +/- 6.4% cells stained positive without IS pretreatment vs 46.1 +/- 3.1% with IS pretreatment, p < 0.001), and this effect was confirmed using the MTT assay. Conclusion: IS has direct cytotoxic effects on PTECs and increases their susceptibility to gentamicin cytotoxicity, and systemic bioaccumulation of IS in CKD could therefore predispose to subsequent AKI following nephrotoxic medication exposure. This phenomenon could be mediated IS-induced downregulation of mitochondrial gene translation associated with dysregulated mitochondrial dynamics and impaired cellular respiration. This study was supported by the George M. O'Brien Kidney Translational Core Center at the University of Michigan (subaward of 5P30-DK081943 14). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aberrant accumulation of NIK promotes tumor growth by dysregulating translation and post-translational modifications in breast cancer

BackgroundIn vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines.MethodsWe established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein.ResultsAs a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1.ConclusionsOur study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.

Protein phosphatase 2A propels follicular T helper cell development in lupus

Follicular helper T (Tfh) cells are important for generating humoral immune responses by helping B cells form germinal centers (GCs) and the production of high-affinity antibodies. However, aberrant Tfh cell expansion also contributes to the generation of self-reactive autoantibodies and promotes autoantibody-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). Protein phosphatase 2A catalytic subunit alpha isoform (PP2A Cα) expression levels are elevated in peripheral T cells of SLE patients and positively correlate with autoantibody titers and disease activity. Here, we demonstrate a critical role of PP2A in Tfh differentiation by using T cell restricted PP2A Cα deficient mice. We observed impaired Tfh differentiation and GC response in two different classical Tfh induction models. Mechanistic studies revealed that downregulation of protein translation of the Tfh lineage transcription factor BCL6 in PP2A deficient T cells. Importantly, we found that PP2A deficiency by either gene knockout or chemical inhibition alleviated lupus severity in mice. Lastly, we confirmed a positive correlation between PP2A Cα and BCL6 protein levels in human CD4+ T cells from patients with SLE. In summary, our study revealed a critical role of PP2A in regulating Tfh cells and suggests it is a potential therapeutic target for lupus.

Intrinsically dysregulated cellular stress signaling genes and gene networks in postpartum depression.

Postpartum depression (PPD) is a leading cause of morbidity and mortality among women. Clinically, the administration and withdrawal of supraphysiologic estradiol and progesterone (E2 + P) can cause affective symptom reoccurrence in women with a history of PPD, but not matched controls. To investigate the cellular basis underlying this differential affective response, lymphoblastoid cell lines (LCLs) were derived from women with and without past PPD and compared transcriptomically in hormone conditions mimicking pregnancy and parturition: supraphysiologic E2 + P-addback; supraphysiologic E2 + P-withdrawal; and no added E2 + P (Baseline). RNA-sequencing identified unique differentially expressed genes (DEGs) in all hormone conditions, but the majority tended to be downregulated in PPD and observed in E2 + P-addback. Two of these DEGs were evolutionarily conserved cellular stress regulators: IMPACT, an integrative response protein maintaining translational homeostasis, and WWTR1, a transcriptional coactivator in the 'Hippo' pathway mediating cell proliferation and survival. Correspondingly, significant gene network modules were linked to cell cycle progression, estrogen response, and immune dysregulation, suggesting innate differences in intracellular signaling in PPD. In certain hormone conditions, PPD LCLs displayed increased GATA3 expression (an upstream regulator of IMPACT and WWTR1) and differentially phosphorylated eiF2α (the ultimate downstream target of IMPACT). Taken together, these transcriptomic data primarily implicate innately dysregulated cellular responses as potentially influencing mood and/or escalating PPD risk. Furthermore, the intrinsic downregulation of IMPACT's translation and WWTR1's transcription networks may suggest a novel link between PPD and a compromised ability to maintain homeostasis in the context of cellular stress occurring during pregnancy and parturition.

(p)ppGpp - an important player during heat shock response.

The alarmones and second messengers (p)ppGpp are important for the cellular response to amino acid starvation. Although the stringent response is present in many bacteria, the targets and functions of (p)ppGpp can differ between species, and our knowledge of (p)ppGpp targets is constantly expanding. Recently, it was demonstrated that these alarmones are also part of the heat shock response in Bacillus subtilis and that there is a functional overlap with the oxidative and heat stress transcriptional regulator Spx. Here, the (p)ppGpp second messenger alarmones allow the fast stress-induced downregulation of translation while Spx inhibits the further expression of translation-related genes to lower the load on the protein quality control system, while the chaperone and protease expression is induced. In this review, we discuss the role of (p)ppGpp and its intricate connections in the complex network of stress sensing, heat shock response, and adaptation in B. subtilis cells.

The global downregulation of protein synthesis observed during hepatogenic maturation is associated with a decrease in TOP mRNA translation.

Translational regulation is of paramount importance for proteome remodeling during stem cell differentiation at both the global and the transcript-specific levels. In this study, we characterized translational remodeling during hepatogenic differentiation of induced pluripotent stem cells (iPSCs) by polysome profiling. We demonstrate that protein synthesis increases during exit from pluripotency and is then globally repressed during later steps of hepatogenic maturation. This global downregulation of translation is accompanied by a decrease in the abundance of protein components of the translation machinery, which involves a global reduction in translational efficiency ofterminal oligopyrimidine tract (TOP) mRNA encoding translation-related factors. Despite global translational repression during hepatogenic differentiation, key hepatogenic genes remain efficiently translated, and the translation of several transcripts involved in hepatospecific functions and metabolic maturation is even induced. We conclude that, during hepatogenic differentiation, a global decrease in protein synthesis is accompanied by a specific translational rewiring of hepatospecific transcripts.

Light-Activated Translation of Different mRNAs in Cells via Wavelength-Dependent Photouncaging.

The 5' cap is a hallmark of eukaryotic mRNA involved in the initiation of translation. Its modification with a single photo-cleavable group can bring translation of mRNA under the control of light. However, UV irradiation causes cell stress and downregulation of translation. Furthermore, complex processes often involve timed expression of more than one gene. The approach would thus greatly benefit from the ability to photo-cleave by blue light and to control more than one mRNA at a time. We report the synthesis of a 5' cap modified with a 7-(diethylamino)coumarin (CouCap) and adapted conditions for in vitro transcription. Translation of the resulting CouCap-mRNA is muted in vitro and in mammalian cells, and can be initiated by irradiation with 450 nm. The native cap is restored and no non-natural residues nor sequence alterations remain in the mRNA. Multiplexing for two different mRNAs was achieved by combining cap analogs with coumarin- and ortho-nitrobenzyl-based photo-cleavable groups.

A critical period of translational control during brain development at codon resolution

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis and products of mRNA translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin-binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome biogenesis, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing activity of eIF4EBP1, a direct inhibitor of ribosome biogenesis, is concurrent with ribosome downregulation and affects neurogenesis of the Satb2 lineage. Thus, the molecular logic of brain development includes the refinement of transcriptional programs by translation. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource at https://shiny.mdc-berlin.de/cortexomics.

Mapping the mutual transcriptional responses during Candida albicans and human macrophage interactions by dual RNA-sequencing

Candida albicans is the leading human fungal pathogen that can cause mucosal and systemic fungal infections. Host phagocytes are the primary immune defense against invading fungal pathogens including C. albicans. To better understand the host-pathogen interaction between C. albicans and host phagocytes, we utilized a human macrophage model of THP-1 macrophages and examined the mutual transcriptomic response of C. albicans and host macrophages by dual RNA-sequencing. Both C. albicans and macrophages displayed marked changes in their transcriptional profiles post 2 h coincubation. We show that C. albicans responds to human macrophages differently than its known response to murine macrophages. C. albicans displays upregulation of its translational machinery and downregulation of glyoxylate and tricarboxylic acid (TCA) cycle upon macrophage phagocytosis. C. albicans triggered strong induction of genes associated with cell surface-mediated signaling and proinflammatory response in THP-1 macrophages. Finally, our data reveal that IL-1β and TNF signaling are central in mounting a proinflammatory response against C. albicans via MAP kinase, and chemokines and cytokines mediated signaling. Overall, current work uncovers the mutual responses of C. albicans and human macrophages towards each other presenting a better understanding of their interaction during C. albicans infections.

DDX60 selectively reduces translation off viral type II internal ribosome entry sites.

Co-opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon-stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5' cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV-1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5' capped mRNA. Our study identifies a novel facet in the repertoire of interferon-stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.

Prostate cancer resistance leads to a global deregulation of translation factors and unconventional translation.

Emerging evidence associates translation factors and regulators to tumorigenesis. However, our understanding of translational changes in cancer resistance is still limited. Here, we generated an enzalutamide-resistant prostate cancer (PCa) model, which recapitulated key features of clinical enzalutamide-resistant PCa. Using this model and poly(ribo)some profiling, we investigated global translation changes that occur during acquisition of PCa resistance. We found that enzalutamide-resistant cells exhibit an overall decrease in mRNA translation with a specific deregulation in the abundance of proteins involved in mitochondrial processes and in translational regulation. However, several mRNAs escape this translational downregulation and are nonetheless bound to heavy polysomes in enzalutamide-resistant cells suggesting active translation. Moreover, expressing these corresponding genes in enzalutamide-sensitive cells promotes resistance to enzalutamide treatment. We also found increased association of long non-coding RNAs (lncRNAs) with heavy polysomes in enzalutamide-resistant cells, suggesting that some lncRNAs are actively translated during enzalutamide resistance. Consistent with these findings, expressing the predicted coding sequences of known lncRNAs JPX, CRNDE and LINC00467 in enzalutamide-sensitive cells drove resistance to enzalutamide. Taken together, this suggests that aberrant translation of specific mRNAs and lncRNAs is a strong indicator of PCa enzalutamide resistance, which points towards novel therapeutic avenues that may target enzalutamide-resistant PCa.

Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature.

The transformation of normal to malignant cells is accompanied by substantial changes in gene expression programs through diverse mechanisms. Here, we examined the changes in the landscape of transcription start sites and alternative promoter (AP) usage and their impact on the translatome in TCL1-driven chronic lymphocytic leukemia (CLL). Our findings revealed a marked elevation of APs in CLL B cells from Eµ-Tcl1 transgenic mice, which are particularly enriched with intra-genic promoters that generate N-terminally truncated or modified proteins. Intra-genic promoter activation is mediated by (1) loss of function of 'closed chromatin' epigenetic regulators due to the generation of inactive N-terminally modified isoforms or reduced expression; (2) upregulation of transcription factors, including c-Myc, targeting the intra-genic promoters and their associated enhancers. Exogenous expression of Tcl1 in MEFs is sufficient to induce intra-genic promoters of epigenetic regulators and promote c-Myc expression. We further found a dramatic translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing metabolic stress. These findings uncovered the role of Tcl1 oncogenic function in altering promoter usage and mRNA translation in leukemogenesis.