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Major depression and anxiety disorders are significant causes of disability and socioeconomic burden. Despite the prevalence and considerable impact of these affective disorders, their pathophysiology remains elusive. Thus, there is an urgent need to develop novel therapeutics for these conditions. We evaluated the role of SIRT1 in regulating dysfunctional processes of reward by using chronic social defeat stress to induce depression- and anxiety-like behaviors. Chronic social defeat stress induces physiological and behavioral changes that recapitulate depression-like symptomatology and alters gene expression programs in the nucleus accumbens, but cell type–specific changes in this critical structure remain largely unknown. We examined transcriptional profiles of D1-expressing medium spiny neurons (MSNs) lacking deacetylase activity of SIRT1 by RNA sequencing in a cell type–specific manner using the RiboTag line of mice. We analyzed differentially expressed genes using gene ontology tools including SynGO and EnrichR and further demonstrated functional changes in D1-MSN–specific SIRT1 knockout (KO) mice using electrophysiological and behavioral measurements. RNA sequencing revealed altered transcriptional profiles of D1-MSNs lacking functional SIRT1 and showed specific changes in synaptic genes including glutamatergic and GABAergic (gamma-aminobutyric acidergic) receptors in D1-MSNs. These molecular changes may be associated with decreased excitatory and increased inhibitory neural activity in Sirt1 KO D1-MSNs, accompanied by morphological changes. Moreover, the D1-MSN–specific Sirt1 KO mice exhibited proresilient changes in anxiety- and depression-like behaviors. SIRT1 coordinates excitatory and inhibitory synaptic genes to regulate the GABAergic output tone of D1-MSNs. These findings reveal a novel signaling pathway that has potential for the development of innovative treatments for affective disorders. [ABSTRACT FROM AUTHOR]

Astrocytes and microglia undergo dynamic and complex morphological and functional changes following ischemic stroke, which are instrumental in both inflammatory responses and neural repair. While gene expression alterations poststroke have been extensively studied, investigations into posttranscriptional regulatory mechanisms, specifically alternative splicing (AS), remain limited. Utilizing previously reported Ribo‐Tag‐seq data, this study analyzed AS alterations in poststroke astrocytes and microglia from young adult male and female mice. Our findings reveal that in astrocytes, compared to the sham group, 109 differential alternative splicing (DAS) events were observed at 4 h poststroke, which increased to 320 at day 3. In microglia, these numbers were 316 and 266, respectively. Interestingly, the disparity between DAS genes and differentially expressed genes is substantial, with fewer than 10 genes shared at both poststroke time points in astrocytes and microglia. Gene ontology enrichment analysis revealed the involvement of these DAS genes in diverse functions, encompassing immune response (Adam8, Ccr1), metabolism (Acsl6, Pcyt2, Myo5a), and developmental cell growth (App), among others. Selective DAS events were further validated by semiquantitative RT‐PCR. Overall, this study comprehensively describes the AS alterations in astrocytes and microglia during the hyperacute and acute phases of ischemic stroke and underscores the significance of certain hub DAS events in neuroinflammatory processes. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that tumor-specific neoantigens are ideal targets for cancer immunotherapy. However, how to predict tumor neoantigens based on translatome data remains obscure. Through the extraction of ribosome-nascent chain complexes (RNCs) from LLC cells, followed by RNC-mRNA extraction, RNC-mRNA sequencing, and comprehensive bioinformatic analysis, we successfully identified proteins undergoing translatome and exhibiting mutations in the cells. Subsequently, novel antigens identification was analyzed by the interaction between their high affinity and the Major Histocompatibility Complex (MHC). Neoantigens immunogenicity was analyzed by enzyme-linked immunospot assay (ELISpot). Finally, in vivo experiments in mice were conducted to evaluate the antitumor effects of translatome-derived neoantigen peptides on lung cancer. The results showed that ten neoantigen peptides were identified and synthesized by translatome data from LLC cells; 8 out of the 10 neoantigens had strong immunogenicity. The neoantigen peptide vaccine group exhibited significant tumor growth inhibition effect. In conclusion, neoantigen peptide vaccine derived from the translatome of lung cancer exhibited significant tumor growth inhibition effect. [ABSTRACT FROM AUTHOR]

Abstract Background Recent studies uncovered pervasive transcription and translation of thousands of noncanonical open reading frames (nORFs) outside of annotated genes. The contribution of nORFs to cellular phenotypes is difficult to infer using conventional approaches because nORFs tend to be short, of recent de novo origins, and lowly expressed. Here we develop a dedicated coexpression analysis framework that accounts for low expression to investigate the transcriptional regulation, evolution, and potential cellular roles of nORFs in Saccharomyces cerevisiae. Results Our results reveal that nORFs tend to be preferentially coexpressed with genes involved in cellular transport or homeostasis but rarely with genes involved in RNA processing. Mechanistically, we discover that young de novo nORFs located downstream of conserved genes tend to leverage their neighbors’ promoters through transcription readthrough, resulting in high coexpression and high expression levels. Transcriptional piggybacking also influences the coexpression profiles of young de novo nORFs located upstream of genes, but to a lesser extent and without detectable impact on expression levels. Transcriptional piggybacking influences, but does not determine, the transcription profiles of de novo nORFs emerging nearby genes. About 40% of nORFs are not strongly coexpressed with any gene but are transcriptionally regulated nonetheless and tend to form entirely new transcription modules. We offer a web browser interface ( https://carvunislab.csb.pitt.edu/shiny/coexpression/ ) to efficiently query, visualize, and download our coexpression inferences. Conclusions Our results suggest that nORF transcription is highly regulated. Our coexpression dataset serves as an unprecedented resource for unraveling how nORFs integrate into cellular networks, contribute to cellular phenotypes, and evolve.

Abstract Background Soybean establishes a mutualistic interaction with nitrogen-fixing rhizobacteria, acquiring most of its nitrogen requirements through symbiotic nitrogen fixation. This crop is susceptible to water deficit; evidence suggests that its nodulation status—whether it is nodulated or not—can influence how it responds to water deficit. The translational control step of gene expression has proven relevant in plants subjected to water deficit. Results Here, we analyzed soybean roots’ differential responses to water deficit at transcriptional, translational, and mixed (transcriptional + translational) levels. Thus, the transcriptome and translatome of four combined-treated soybean roots were analyzed. We found hormone metabolism-related genes among the differentially expressed genes (DEGs) at the translatome level in nodulated and water-restricted plants. Also, weighted gene co-expression network analysis followed by differential expression analysis identified gene modules associated with nodulation and water deficit conditions. Protein-protein interaction network analysis was performed for subsets of mixed DEGs of the modules associated with the plant responses to nodulation, water deficit, or their combination. Conclusions Our research reveals that the stand-out processes and pathways in the before-mentioned plant responses partially differ; terms related to glutathione metabolism and hormone signal transduction (2 C protein phosphatases) were associated with the response to water deficit, terms related to transmembrane transport, response to abscisic acid, pigment metabolic process were associated with the response to nodulation plus water deficit. Still, two processes were common: galactose metabolism and branched-chain amino acid catabolism. A comprehensive analysis of these processes could lead to identifying new sources of tolerance to drought in soybean.

Introduction: The unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement. Objectives: Despite advances in research, the impact of post-transcriptional regulation on fiber development and translatome profiling at different stages of fiber growth in cotton (G. hirsutum) remains unexplored. Methods: We utilized a combination of reference-guided de novo transcriptome assembly and ribosome profiling techniques to uncover the hidden mechanisms of translational control in eight distinct tissues of upland cotton. Results: Our study identified P-site distribution at three-nucleotide periodicity and dominant ribosome footprint at 27 nucleotides. Specifically, we have detected 1,589 small open reading frames (sORFs), including 1,376 upstream ORFs (uORFs) and 213 downstream ORFs (dORFs), as well as 552 long non-coding RNAs (lncRNAs) with potential coding functions, which fine-tune the annotation of the cotton genome. Further, we have identified novel genes and lncRNAs with strong translation efficiency (TE), while sORFs were found to affect mRNA transcription levels during fiber elongation. The reliability of these findings was confirmed by the high consistency in correlation and synergetic fold change between RNA-sequencing (RNA-seq) and Ribosome-sequencing (Ribo-seq) analyses. Additionally, integrated omics analysis of the normal fiber ZM24 and short fiber pag1 cotton mutant revealed several differentially expressed genes (DEGs), and fiber-specific expressed (high/low) genes associated with sORFs (uORFs and dORFs). These findings were further supported by the overexpression and knockdown of GhKCS6, a gene associated with sORFs in cotton, and demonstrated the potential regulation of the mechanism governing fiber elongation on both the transcriptional and post-transcriptional levels. Conclusion: Reference-guided transcriptome assembly and the identification of novel transcripts fine-tune the annotation of the cotton genome and predicted the landscape of fiber development. Our approach provided a high-throughput method, based on multi-omics, for discovering unannotated ORFs, hidden translational control, and complex regulatory mechanisms in crop plants.

Background: Soybean establishes a mutualistic interaction with nitrogen-fixing rhizobacteria, acquiring most of its nitrogen requirements through symbiotic nitrogen fixation. This crop is susceptible to water deficit; evidence suggests that its nodulation status—whether it is nodulated or not—can influence how it responds to water deficit. The translational control step of gene expression has proven relevant in plants subjected to water deficit. Results: Here, we analyzed soybean roots' differential responses to water deficit at transcriptional, translational, and mixed (transcriptional + translational) levels. Thus, the transcriptome and translatome of four combined-treated soybean roots were analyzed. We found hormone metabolism-related genes among the differentially expressed genes (DEGs) at the translatome level in nodulated and water-restricted plants. Also, weighted gene co-expression network analysis followed by differential expression analysis identified gene modules associated with nodulation and water deficit conditions. Protein-protein interaction network analysis was performed for subsets of mixed DEGs of the modules associated with the plant responses to nodulation, water deficit, or their combination. Conclusions: Our research reveals that the stand-out processes and pathways in the before-mentioned plant responses partially differ; terms related to glutathione metabolism and hormone signal transduction (2 C protein phosphatases) were associated with the response to water deficit, terms related to transmembrane transport, response to abscisic acid, pigment metabolic process were associated with the response to nodulation plus water deficit. Still, two processes were common: galactose metabolism and branched-chain amino acid catabolism. A comprehensive analysis of these processes could lead to identifying new sources of tolerance to drought in soybean. [ABSTRACT FROM AUTHOR]

Aims Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue. Methods and results To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr−/−, ApoB100/100 to generate SMCTRAP−AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip , Lum , Mfge8 , Spp1 , and Serpina3 , which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4 , Cd276 , inter-alpha-trypsin inhibitor-4 (Itih4), Myof , Pcdh11x , Rab31 , Serpinb6b , Slc35e4 , Slc8a3 , and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals. Conclusion We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality. [ABSTRACT FROM AUTHOR]

Dark–light and light–dark transitions during the day are switching points of leaf metabolism that strongly affect the regulatory state of the cells, and this change is hypothesized to affect the translatome. The cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1 and GAPC2 function in glycolysis, and carbohydrate and energy metabolism, but GAPC1/C2 also shows moonlighting functions in gene expression and post-transcriptional regulation. In this study we examined the rapid reprogramming of the translatome that occurs within 10 min at the end of the night and the end of the day in wild-type (WT) Arabidopsis and a gapc1/c2 double-knockdown mutant. Metabolite profiling compared to the WT showed that gapc1/c2 knockdown led to increases in a set of metabolites at the start of day, particularly intermediates of the citric acid cycle and linked pathways. Differences in metabolite changes were also detected at the end of the day. Only small sets of transcripts changed in the total RNA pool; however, RNA-sequencing revealed major alterations in polysome-associated transcripts at the light-transition points. The most pronounced difference between the WT and gapc1/c2 was seen in the reorganization of the translatome at the start of the night. Our results are in line with the proposed hypothesis that GAPC1/C2 play a role in the control of the translatome during light/dark transitions. [ABSTRACT FROM AUTHOR]

[Display omitted] • The transcriptome and translatome of 8 tissues of cotton generated large datasets. • De novo transcriptome assembly and ribosome profiling fine-tune cotton genome annotation. • Unannotated translational events including sORFs (uORFs and dORF), and lncRNAs were identified. • Omics integrated analysis of normal fiber ZM24 and short fiber pag1 explored fiber-specific genes. • GhKCS6 overexpression showed greater fiber length than the control and validated our approach. The unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement. Despite advances in research, the impact of post-transcriptional regulation on fiber development and translatome profiling at different stages of fiber growth in cotton (G. hirsutum) remains unexplored. We utilized a combination of reference-guided de novo transcriptome assembly and ribosome profiling techniques to uncover the hidden mechanisms of translational control in eight distinct tissues of upland cotton. Our study identified P-site distribution at three-nucleotide periodicity and dominant ribosome footprint at 27 nucleotides. Specifically, we have detected 1,589 small open reading frames (sORFs), including 1,376 upstream ORFs (uORFs) and 213 downstream ORFs (dORFs), as well as 552 long non-coding RNAs (lncRNAs) with potential coding functions, which fine-tune the annotation of the cotton genome. Further, we have identified novel genes and lncRNAs with strong translation efficiency (TE), while sORFs were found to affect mRNA transcription levels during fiber elongation. The reliability of these findings was confirmed by the high consistency in correlation and synergetic fold change between RNA-sequencing (RNA-seq) and Ribosome-sequencing (Ribo-seq) analyses. Additionally, integrated omics analysis of the normal fiber ZM24 and short fiber pag1 cotton mutant revealed several differentially expressed genes (DEGs), and fiber-specific expressed (high/low) genes associated with sORFs (uORFs and dORFs). These findings were further supported by the overexpression and knockdown of GhKCS6 , a gene associated with sORFs in cotton, and demonstrated the potential regulation of the mechanism governing fiber elongation on both the transcriptional and post-transcriptional levels. Reference-guided transcriptome assembly and the identification of novel transcripts fine-tune the annotation of the cotton genome and predicted the landscape of fiber development. Our approach provided a high-throughput method, based on multi-omics, for discovering unannotated ORFs, hidden translational control, and complex regulatory mechanisms in crop plants. [ABSTRACT FROM AUTHOR]

Abstract Leguminous plants can establish endosymbiotic relationships with nitrogen-fixing soil rhizobacteria. Bacterial infection and nodule organogenesis are two independent but highly coordinated genetic programs that are active during this interaction. These genetic programs can be regulated along all the stages of gene expression. Most of the studies, for both eukaryotes and prokaryotes, focused on the transcriptional regulation level determining the abundance of mRNAs. However, it has been demonstrated that mRNA levels only sometimes correlate with the abundance or activity of the coded proteins. For this reason, in the past two decades, interest in the role of translational control of gene expression has increased, since the subset of mRNA being actively translated outperforms the information gained only by the transcriptome. In the case of legume-rhizobia interactions, the study of the translatome still needs to be explored further. Therefore, this review aims to discuss the methodologies for analyzing polysome-associated mRNAs at the genome-scale and their contribution to studying translational control to understand the complexity of this symbiotic interaction. Moreover, the Dual RNA-seq approach is discussed for its relevance in the context of a symbiotic nodule, where intricate multi-species gene expression networks occur.

Exposure to anesthesia in early life may cause severe damage to the brain and lead to cognitive impairment. The underlying mechanisms, which have only been investigated in a limited scale, remains largely elusive. We performed translatome and transcriptome sequencing together for the first time in hippocampus of neonatal mice that were exposed to sevoflurane. We treated a group of neonatal mice with 2.5 % sevoflurane for 2 h on day 6, 7, 8, 9 and treated another group on day 6, 7. We performed behavioral study after day 30 for both groups and the control to evaluate the cognitive impairment. On day 36, we collected translatome and transcriptome from the hippocampus in the two groups, compared the gene expression levels between the groups and the control, and validated the results with RT-qPCR. We identified 1750 differentially expressed genes (DEGs) from translatome comparison and 1109 DEGs from transcriptome comparison. As expected, translatome-based DEGs significantly overlapped with transcriptome-based DEGs, and functional enrichment analysis generated similar enriched cognition-related GO terms and KEGG pathways. However, for many genes like Hspa5, their alterations in translatome differed remarkably from those in transcriptome, and Western blot results were largely concordant with the former, suggesting that translational regulation plays a significant role in cellular response to sevoflurane. Our study revealed global alterations in translatome and transcriptome of mice hippocampus after neonatal exposure to sevoflurane anesthesia and highlighted the importance of translatome analysis in understanding the mechanisms responsible for anesthesia-induced cognitive impairment.

Abstract Background Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. Methods Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. Results Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. Conclusions Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

Long noncoding RNAs (lncRNAs) have been extensively identified in eukaryotic genomes and have been shown to play critical roles in the development of multiple cancers. Through the application and development of ribosome analysis and sequencing technologies, advanced studies have discovered the translation of lncRNAs. Although lncRNAs were originally defined as noncoding RNAs, many lncRNAs actually contain small open reading frames that are translated into peptides. This opens a broad area for the functional investigation of lncRNAs. Here, we introduce prospective methods and databases for screening lncRNAs with functional polypeptides. We also summarize the specific lncRNA-encoded proteins and their molecular mechanisms that promote or inhibit cancerous. Importantly, the role of lncRNA-encoded peptides/proteins holds promise in cancer research, but some potential challenges remain unresolved. This review includes reports on lncRNA-encoded peptides or proteins in cancer, aiming to provide theoretical basis and related references to facilitate the discovery of more functional peptides encoded by lncRNA, and to further develop new anti-cancer therapeutic targets as well as clinical biomarkers of diagnosis and prognosis. [ABSTRACT FROM AUTHOR]

Background: Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. Methods: Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. Results: Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. Conclusions: Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND. [ABSTRACT FROM AUTHOR]

Abstract Background The oocyte-to-embryo transition (OET) converts terminally differentiated gametes into a totipotent embryo and is critically controlled by maternal mRNAs and proteins, while the genome is silent until zygotic genome activation. How the transcriptome, translatome, and proteome are coordinated during this critical developmental window remains poorly understood. Results Utilizing a highly sensitive and quantitative mass spectrometry approach, we obtain high-quality proteome data spanning seven mouse stages, from full-grown oocyte (FGO) to blastocyst, using 100 oocytes/embryos at each stage. Integrative analyses reveal distinct proteome reprogramming compared to that of the transcriptome or translatome. FGO to 8-cell proteomes are dominated by FGO-stockpiled proteins, while the transcriptome and translatome are more dynamic. FGO-originated proteins frequently persist to blastocyst while corresponding transcripts are already downregulated or decayed. Improved concordance between protein and translation or transcription is observed for genes starting translation upon meiotic resumption, as well as those transcribed and translated only in embryos. Concordance between protein and transcription/translation is also observed for proteins with short half-lives. We built a kinetic model that predicts protein dynamics by incorporating both initial protein abundance in FGOs and translation kinetics across developmental stages. Conclusions Through integrative analyses of datasets generated by ultrasensitive methods, our study reveals that the proteome shows distinct dynamics compared to the translatome and transcriptome during mouse OET. We propose that the remarkably stable oocyte-originated proteome may help save resources to accommodate the demanding needs of growing embryos. This study will advance our understanding of mammalian OET and the fundamental principles governing gene expression.

Protozoan parasites are known for their remarkable capacity to persist within the bodies of vertebrate hosts, which frequently results in prolonged infections and the recurrence of diseases. Understanding the molecular mechanisms that underlie the event of persistence is of paramount significance to develop innovative therapeutic approaches, given that these pathways still need to be thoroughly elucidated. The present article provides a comprehensive overview of the latest developments in the investigation of protozoan persistence in vertebrate hosts. The focus is primarily on the function of persisters, their formation within the host, and the specific molecular interactions between host and parasite while they persist. Additionally, we examine the metabolomic, transcriptional, and translational changes that protozoan parasites undergo during persistence within vertebrate hosts, focusing on major parasites such as Plasmodium spp., Trypanosoma spp., Leishmania spp., and Toxoplasma spp. Key findings of our study suggest that protozoan parasites deploy several molecular and physiological strategies to evade the host immune surveillance and sustain their persistence. Furthermore, some parasites undergo stage differentiation, enabling them to acclimate to varying host environments and immune challenges. More often, stressors such as drug exposure were demonstrated to impact the formation of protozoan persisters significantly. Understanding the molecular mechanisms regulating the persistence of protozoan parasites in vertebrate hosts can reinvigorate our current insights into host–parasite interactions and facilitate the development of more efficacious disease therapeutics. [ABSTRACT FROM AUTHOR]

Abstract Background Maize (Zea mays L.) is one of the most important crops worldwide. Although sophisticated maize gene regulatory networks (GRNs) have been constructed for functional genomics and phenotypic dissection, a multi-omics GRN connecting the translatome and transcriptome is lacking, hampering our understanding and exploration of the maize regulatome. Results We collect spatio-temporal translatome and transcriptome data and systematically explore the landscape of gene transcription and translation across 33 tissues or developmental stages of maize. Using this comprehensive transcriptome and translatome atlas, we construct a multi-omics GRN integrating mRNAs and translated mRNAs, demonstrating that translatome-related GRNs outperform GRNs solely using transcriptomic data and inter-omics GRNs outperform intra-omics GRNs in most cases. With the aid of the multi-omics GRN, we reconcile some known regulatory networks. We identify a novel transcription factor, ZmGRF6, which is associated with growth. Furthermore, we characterize a function related to drought response for the classic transcription factor ZmMYB31. Conclusions Our findings provide insights into spatio-temporal changes across maize development at both the transcriptome and translatome levels. Multi-omics GRNs represent a useful resource for dissection of the regulatory mechanisms underlying phenotypic variation.

BackgroundCopper (Cu) is an essential metal mediating a variety of vital biological reactions with its redox property. Its dyshomeostasis has been associated with accelerated cognitive decline and neurodegenerative disorders, such as Alzheimer's disease (AD). However, underlying neurotoxic mechanisms elicited by dysregulated Cu remain largely elusive. We and others previously demonstrated that exposure to Cu in drinking water significantly exacerbated pathological hallmarks of AD and pro-inflammatory activation of microglia, coupled with impaired phagocytic capacity, in mouse models of AD.MethodsIn the present study, we extended our investigation to evaluate whether chronic Cu exposure to wild-type (WT) and J20 mouse model of AD perturbs homeostatic dynamics of microglia and contributes to accelerated transformation of microglia towards degenerative phenotypes that are closely associated with neurodegeneration. We further looked for evidence of alterations in the microglial morphology and spatial memory of the Cu-exposed mice to assess the extent of the Cu toxicity.ResultsWe find that chronic Cu exposure to pre-pathological J20 mice upregulates the translation of degenerative genes and represses homeostatic genes within microglia even in the absence amyloid-beta plaques. We also observe similar expression signatures in Cu-exposed WT mice, suggesting that excess Cu exposure alone could lead to perturbed microglial homeostatic phenotypes and contribute to accelerated cognitive decline.ConclusionOur findings highlight the risk of chronic Cu exposure on cognitive decline and altered microglia activation towards degenerative phenotypes. These changes may represent one of the key mechanisms linking Cu exposure or its dyshomeostasis to an increased risk for AD.

Key early features of amyotrophic lateral sclerosis (ALS) are denervation of neuromuscular junctions and axonal degeneration. Motor neuron homeostasis relies on local translation through controlled regulation of axonal mRNA localization, transport, and stability. Yet the composition of the local transcriptome, translatome (mRNAs locally translated), and proteome during health and disease remains largely unexplored. This review covers recent discoveries on axonal translation as a critical mechanism for neuronal maintenance/survival. We focus on two RNA binding proteins, transactive response DNA binding protein-43 (TDP-43) and fused in sarcoma (FUS), whose mutations cause ALS and frontotemporal dementia (FTD). Emerging evidence points to their essential role in the maintenance of axons and synapses, including mRNA localization, transport, and local translation, and whose dysfunction may contribute to ALS. Finally, we describe recent advances in omics-based approaches mapping compartment-specific local RNA and protein compositions, which will be invaluable to elucidate fundamental local processes and identify key targets for therapy development. Piol et al. review key breakthroughs made on the role of local translation in the maintenance of axons and neuromuscular junctions and their demise in TDP-43- and FUS-mediated ALS, further discussing spatial omics as promising technologies to study the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly wellcoordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3'-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases. [ABSTRACT FROM AUTHOR]

Understanding the impact of elevated CO2 (eCO2 ) in global agriculture is important given climate change projections. Breeding climate-resilient crops depends on genetic variation within naturally varying populations. The effect of genetic variation in response to eCO2 is poorly understood, especially in crop species. We describe the different ways in which Solanum lycopersicum and its wild relative S. pennellii respond to eCO2 , from cell anatomy, to the transcriptome, and metabolome. We further validate the importance of translational regulation as a potential mechanism for plants to adaptively respond to rising levels of atmospheric CO2 .

Abstract Background Ribosome profiling, also known as Ribo-seq, is a powerful technique to study genome-wide mRNA translation. It reveals the precise positions and quantification of ribosomes on mRNAs through deep sequencing of ribosome footprints. We previously optimized the resolution of this technique in plants. However, several key reagents in our original method have been discontinued, and thus, there is an urgent need to establish an alternative protocol. Results Here we describe a step-by-step protocol that combines our optimized ribosome footprinting in plants with available custom library construction methods established in yeast and bacteria. We tested this protocol in 7-day-old Arabidopsis seedlings and evaluated the quality of the sequencing data regarding ribosome footprint length, mapped genomic features, and the periodic properties corresponding to actively translating ribosomes through open resource bioinformatic tools. We successfully generated high-quality Ribo-seq data comparable with our original method. Conclusions We established a custom library construction method for super-resolution Ribo-seq in Arabidopsis. The experimental protocol and bioinformatic pipeline should be readily applicable to other plant tissues and species.

MAIT cells are multifunctional innate-like effector cells recognizing bacterialderived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-typespecifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1β, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages. [ABSTRACT FROM AUTHOR]

Matrix-deprivation stress leads to cell-death by anoikis, whereas overcoming anoikis is critical for cancer metastasis. Work from our lab and others has identified a crucial role for the cellular energy sensor AMPK in anoikis-resistance, highlighting a key role for metabolic reprogramming in stress survival. Protein synthesis is a major energy-consuming process that is tightly regulated under stress. Although an increase in protein synthesis in AMPK-depleted experimentally-transformed MEFs has been associated with anoikis, the status and regulation of protein translation in epithelial-origin cancer cells facing matrix-detachment remains largely unknown. Our study shows that protein translation is mechanistically abrogated at both initiation and elongation stages by the activation of the unfolded protein response (UPR) pathway and inactivation of elongation factor eEF2, respectively. Additionally, we show inhibition of the mTORC1 pathway known for regulation of canonical protein synthesis. We further functionally assay this inhibition using SUnSET assay, which demonstrates repression of global protein synthesis in MDA-MB-231 and MCF7 breast cancer cells when subjected to matrix-deprivation. In order to gauge the translational status of matrix-deprived cancer cells, we undertook polysome profiling. Our data revealed reduced but continuous mRNA translation under matrix-deprivation stress. An integrated analysis of transcriptomic and proteomic data further identifies novel targets that may aid cellular adaptations to matrix-deprivation stress and can be explored for therapeutic intervention.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by the progressive loss of nigrostriatal dopaminergic neurons (DANs), involving the dysregulation of both neurons and glial cells. Cell type- and region-specific gene expression profiles can provide an effective source for revealing the mechanisms of PD. In this study, we adopted the RiboTag approach to obtain cell type (DAN, microglia, astrocytes)- and brain region (substantia nigra, caudate-putamen)-specific translatomes at an early stage in an MPTP-induced mouse model of PD. Through DAN-specific translatome analysis, the glycosphingolipid biosynthetic process was identified as a significantly downregulated pathway in the MPTP-treated mice. ST8Sia6, a key downregulated gene related to glycosphingolipid biosynthesis, was confirmed to be downregulated in nigral DANs from postmortem brains of patients with PD. Specific expression of ST8Sia6 in DANs exerts anti-inflammatory and neuroprotective effects in MPTP-treated mice. Through cell type (microglia vs. astrocyte) and brain region (substantia nigra vs. caudate-putamen) comparisons, nigral microglia showed the most intense immune responses. Microglia and astrocytes in the substantia nigra showed similar levels of activation in interferon-related pathways and interferon gamma (IFNG) was identified as the top upstream regulator in both cell types. This work highlights that the glycosphingolipid metabolism pathway in the DAN is involved in neuroinflammation and neurodegeneration in an MPTP mouse model of PD and provides a new data source for elucidating the pathogenesis of PD.

Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly well-coordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3′-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases.

MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1β, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

Protein is an important component of cells and the main undertaker of life activities. Its synthesis highly relies on complex and precise translation systems. Previous studies have shown that the correlation between mRNA expression and protein expression level is low. One of important factors is that mRNA is not translated into protein at the same rate. Translation rate is the key to determining protein expression, and also affects protein structure and function. Therefore, on the basis of summarizing the mechanism of mRNA translation in eukaryotic and prokaryotic cells, this study further summarized the main factors affecting translation rate and the main technical means of studying translation and translation rate, aiming to provide reference value for the study of translation mechanism in vivo in the future. [ABSTRACT FROM AUTHOR]

The present study aimed to better understand the possibility of utilizing all-trans retinoic acids (ATRA) in acute myeloid leukemia (AML). We found that ATRA significantly suppressed global translation and protein synthesis in AML cells. The efficacy of ATRA in treating AML required its translational regulatory functions, as shown by the fact that the decrease in the universal eukaryotic initiation factor 4E (eIF4E) was essential to maintain the induction of cell growth arrest and differentiation by ATRA. By establishing a specific translational landscape, we suggested that transcripts with simple 5′UTR gained a translational advantage in AML cells during ATRA stress. Based on that, the genes translationally regulated by ATRA were mainly enriched in phosphatidylinositol-3-kinase/Akt (PI3K/AKT) signaling; we subsequently revealed that PI3K/AKT activation was required for ATRA to effectively induce AML cell differentiation. However, PI3K/AKT has been reported to promote the stemness of AML cells. As such, we further suggested that sequential treatment including ATRA and PI3K/AKT inhibitor induced robust apoptosis, extremely inhibited the clonality of AML cells, and suppressed the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-driven transformation of CD34+ hematopoietic stem/progenitor cells. Future clinical studies are warranted to further support the clinical application of the sequential strategy for the effective treatment of AML. [ABSTRACT FROM AUTHOR]

Background: Ribosome profiling, also known as Ribo-seq, is a powerful technique to study genome-wide mRNA translation. It reveals the precise positions and quantification of ribosomes on mRNAs through deep sequencing of ribosome footprints. We previously optimized the resolution of this technique in plants. However, several key reagents in our original method have been discontinued, and thus, there is an urgent need to establish an alternative protocol. Results: Here we describe a step-by-step protocol that combines our optimized ribosome footprinting in plants with available custom library construction methods established in yeast and bacteria. We tested this protocol in 7-day-old Arabidopsis seedlings and evaluated the quality of the sequencing data regarding ribosome footprint length, mapped genomic features, and the periodic properties corresponding to actively translating ribosomes through open resource bioinformatic tools. We successfully generated high-quality Ribo-seq data comparable with our original method. Conclusions: We established a custom library construction method for super-resolution Ribo-seq in Arabidopsis. The experimental protocol and bioinformatic pipeline should be readily applicable to other plant tissues and species. [ABSTRACT FROM AUTHOR]

Heterosis, the phenomenon in which hybrids outperform their parents, has been utilized in maize (Zea mays L.) for over 100 years. To provide a more complete understanding of heterosis, we collected a comprehensive transcriptome and translatome dataset on seedling leaves for B73, Mo17, and their F1 hybrid, which provided a dynamic landscape of transcriptomic and translatomic variation in maize. Although additivity accounted for a large proportion of variation at two omics-levels, an elevated nonadditive effect was observed in the translatome, especially in the translated subgenome maize1 genes, and the genes that switched from additivity in the transcriptome to nonadditivity in the translatome were significantly enriched in the subgenome maize1. Many genes with allele-specific expression and translation show dramatic regulatory switches between the transcriptome and translatome, and partial genes with allele-specific translation underlying regulatory mechanism also exhibited subgenome bias. Interestingly, we found the translated isoforms show different expression patterns compared with transcriptome and more genes changed their dominant isoforms during the genetic flow from parents to the hybrid at the translatome level. The translated genes with switched dominant isoforms significantly biased to the subgenome maize2 while genes with conserved dominant isoforms significantly enriched in subgenome maize1. Together, the dynamic changed patterns in translatome across hybrid and parental lines show translational fractionation of the maize subgenomes, which may be associated with heterosis in maize and provides a potential theoretical basis for breeding.

Protozoan parasites are known for their remarkable capacity to persist within the bodies of vertebrate hosts, which frequently results in prolonged infections and the recurrence of diseases. Understanding the molecular mechanisms that underlie the event of persistence is of paramount significance to develop innovative therapeutic approaches, given that these pathways still need to be thoroughly elucidated. The present article provides a comprehensive overview of the latest developments in the investigation of protozoan persistence in vertebrate hosts. The focus is primarily on the function of persisters, their formation within the host, and the specific molecular interactions between host and parasite while they persist. Additionally, we examine the metabolomic, transcriptional, and translational changes that protozoan parasites undergo during persistence within vertebrate hosts, focusing on major parasites such as Plasmodium spp., Trypanosoma spp., Leishmania spp., and Toxoplasma spp. Key findings of our study suggest that protozoan parasites deploy several molecular and physiological strategies to evade the host immune surveillance and sustain their persistence. Furthermore, some parasites undergo stage differentiation, enabling them to acclimate to varying host environments and immune challenges. More often, stressors such as drug exposure were demonstrated to impact the formation of protozoan persisters significantly. Understanding the molecular mechanisms regulating the persistence of protozoan parasites in vertebrate hosts can reinvigorate our current insights into host–parasite interactions and facilitate the development of more efficacious disease therapeutics.

The four combined treatments were nodulated (N) water-restricted (WR) plants (N+WR), nodulated well-watered (WW) plants (N+WW), non-nodulated (NN) water-restricted plants (NN+WR), and non-nodulated well-watered plants (NN+WW). Graph: Figure 4 Differentially expressed gene (DEG) analysis and GmTrx and GmGrx expression profiles in nodulated (N) and water-restricted (WR) plants with respect to well-watered (WW) and non-nodulated (NN) plants. Graph: Figure 6 String analysis [[41]] derived protein-protein interaction network obtained for the differentially expressed genes down-regulated at the TOTAL+PAR level in nodulated and water-restricted plants with respect to non-nodulated and water-restricted plants (N+WR vs. To gain more insight into the functionality of the DEGs, we analyzed the distribution and localization of the most frequent I GmTrx i and I GmGrx i CREs (stress response: ARE and WUN motif; site-binding-related: MYC and MYB; hormone response: CGTCA, TGACG, TCA, and ABRE; and light response: TCT and Box IV) in the promoter regions of the DEGs. From the total detected I GmTrx i and I GmGrx i genes in our RNA-seq experiment, 10% and 20%, respectively, were differentially expressed in nodulated and water-restricted plants, suggesting a relevant role of these proteins in the plant response to water-deficit stress in nodulation conditions. [Extracted from the article]

Background: Recent studies uncovered pervasive transcription and translation of thousands of noncanonical open reading frames (nORFs) outside of annotated genes. The contribution of nORFs to cellular phenotypes is difficult to infer using conventional approaches because nORFs tend to be short, of recent de novo origins, and lowly expressed. Here we develop a dedicated coexpression analysis framework that accounts for low expression to investigate the transcriptional regulation, evolution, and potential cellular roles of nORFs in Saccharomyces cerevisiae., Results: Our results reveal that nORFs tend to be preferentially coexpressed with genes involved in cellular transport or homeostasis but rarely with genes involved in RNA processing. Mechanistically, we discover that young de novo nORFs located downstream of conserved genes tend to leverage their neighbors' promoters through transcription readthrough, resulting in high coexpression and high expression levels. Transcriptional piggybacking also influences the coexpression profiles of young de novo nORFs located upstream of genes, but to a lesser extent and without detectable impact on expression levels. Transcriptional piggybacking influences, but does not determine, the transcription profiles of de novo nORFs emerging nearby genes. About 40% of nORFs are not strongly coexpressed with any gene but are transcriptionally regulated nonetheless and tend to form entirely new transcription modules. We offer a web browser interface ( https://carvunislab.csb.pitt.edu/shiny/coexpression/ ) to efficiently query, visualize, and download our coexpression inferences., Conclusions: Our results suggest that nORF transcription is highly regulated. Our coexpression dataset serves as an unprecedented resource for unraveling how nORFs integrate into cellular networks, contribute to cellular phenotypes, and evolve., (© 2024. The Author(s).)

The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify all proteins encoded by the human genome. Currently, the human proteome still contains approximately 2000 PE2-PE5 proteins, referring to annotated coding genes that lack sufficient protein-level evidence. During the past 10 years, it has been increasingly difficult to identify PE2-PE5 proteins in C-HPP approaches due to the limited occurrence. Therefore, we proposed that reanalyzing massive MS data sets in repository with newly developed algorithms may increase the occurrence of the peptides of these proteins. In this study, we downloaded 1000 MS data sets via the ProteomeXchange database. Using pFind software, we identified peptides referring to 1788 PE2-PE5 proteins. Among them, 11 PE2 and 16 PE5 proteins were identified with at least 2 peptides, and 12 of them were identified using 2 peptides in a single data set, following the criteria of the HPP guidelines. We found translation evidence for 16 of the 11 PE2 and 16 PE5 proteins in our RNC-seq data, supporting their existence. The properties of the PE2 and PE5 proteins were similar to those of the PE1 proteins. Our approach demonstrated that mining PE2 and PE5 proteins in massive data repository is still worthy, and multidata set peptide identifications may support the presence of PE2 and PE5 proteins or at least prompt additional studies for validation. Extremely high throughput could be a solution to finding more PE2 and PE5 proteins.

Aging-associated muscle wasting is regulated by multiple molecular processes, whereby aberrant mRNA processing regulation induces muscle wasting. The poly(A)-binding protein nuclear 1 (PABPN1) regulates polyadenylation site (PAS) utilization, in the absence of PABPN1 the alternative polyadenylation (APA) is utilized. Reduced PABPN1 levels induce muscle wasting where the expression of cellular processes regulating protein homeostasis, the ubiquitin-proteasome system, and translation, are robustly dysregulated. Translation is affected by mRNA levels, but PABPN1 impact on translation is not fully understood. Here we show that a persistent reduction in PABPN1 levels led to a significant loss of translation efficiency. RNA-sequencing of rRNA-depleted libraries from polysome traces revealed reduced mRNA abundance across ribosomal fractions, as well as reduced levels of small RNAs. We show that the abundance of translated mRNAs in the polysomes correlated with PAS switches at the 3'-UTR. Those mRNAs are enriched in cellular processes that are essential for proper muscle function. This study suggests that the effect of PABPN1 on translation efficiency impacts protein homeostasis in aging-associated muscle atrophy. [ABSTRACT FROM AUTHOR]

Summary: The phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion, and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. Using specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots and further targeted the translatome of newly developed phellem cells using translating ribosome affinity purification followed by mRNA sequencing (TRAP‐SEQ). We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation related to suberin monomer transport and assembly as well as novel transcription regulators were identified. This strategy provided an unprecedented resolution of the translatome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms acting on cell differentiation in periderm tissues of the model plant Arabidopsis. Significance Statement: To improve our understanding of phellem differentiation into a suberized protective layer, we followed the establishment of periderm in Arabidopsis roots and sequenced the phellem‐associated translatome. We found that phellem suberization occurs shortly after pericycle cell divisions with the induction of pivotal suberin biosynthesis genes. In parallel, we detected the activation of three central genetic modules acting throughout phellem differentiation. This study provides a unique and targeted genetic resource for further functional studies of phellem tissues. [ABSTRACT FROM AUTHOR]

Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of a GFP-tagged version of the large subunit ribosomal protein L10a (EGFP-Rpl10a), we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling.

In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.

We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium - resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae , and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton. [ABSTRACT FROM AUTHOR]

To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/. Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength. [ABSTRACT FROM AUTHOR]

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a multitude of complications. Increasing evidence shows that the dietary supplement with betaine, a natural chemical molecule, can effectively reduce the fat accumulation in the liver. Translational regulation is considered to play a vital role in gene expression, but whether betaine functions through the regulation of gene translational level is still unclear. To this end, RNC-seq (mRNAs bound to ribosome-nascent chain complex sequencing) and RNA-seq co-analyses were performed to identify betaine target genes by using the liver samples from high-fat diet adding betaine treated and high-fat diet treated mice. The results showed that betaine does play a lipid-lowering role by regulating the expression of gene translation levels; some NAFLD- and lipid metabolism-associated genes were differentially expressed at translational level, for example. And the translation ratio (TR) of gene significantly increased after betaine treatment. Finally, we identified a novel function gene, Gpc1, which may mediate the lipid-lowering effect of betaine in the liver. To sum up, this study depicted the molecular portrait of mice liver with or without betaine treatment from the angel of translatome and transcriptome, giving insights into the molecular mechanism of betaine-mediated lipid-lowering effect and also providing new clues for understanding and prevention of NAFLD. • Our study provides a study case for the application of translational omics techniques to food and health. • Betaine does play a lipid-lowering role by regulating the expression of gene translation levels. • The translation ratio of gene significantly increased after betaine treatment. • We identified a novel function gene, Gpc1, which mediates the lipid-lowering effect of betaine in the liver. [ABSTRACT FROM AUTHOR]

We investigated the gene-expression variation among humans by analysing previously published mRNA-seq and ribosome footprint profiling of heart left-ventricles from healthy donors. We ranked the genes according to their coefficient of variation values and found that the top 5% most variable genes had special features compared to the rest of the genome, such as lower mRNA levels and shorter half-lives coupled to increased translation efficiency. We observed that these genes are mostly involved with immune response and have a pleiotropic effect on disease phenotypes, indicating that asymptomatic conditions contribute to the gene expression diversity of healthy individuals. [ABSTRACT FROM AUTHOR]

ABSTRACT Cyanobacteria are promising industrial platforms owing to their ability to produce diverse natural secondary metabolites and nonnative value-added biochemicals from CO2 and light. To fully utilize their industrial potency, it is critical to understand their photosynthetic efficiency under various environmental conditions. In this study, we elucidated the inhibitory mechanisms of photosynthesis under high-light and low-temperature stress conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Under each stress condition, the transcript abundance and translation efficiency were measured using transcriptome sequencing (RNA-seq) and ribosome profiling, and the genome-wide transcription unit architecture was constructed by data integration of transcription start sites and transcript 3′-end positions obtained from differential RNA-seq and sequencing of 3′-ends (Term-seq), respectively. Our results suggested that the mode of photosynthesis inhibition differed between the two stress conditions; high light stress induced photodamage responses, while low temperature stress impaired the translation efficiency of photosynthesis-associated genes. In particular, poor translation of photosystem I resulted from ribosome stalling at the untranslated regions, affecting the overall photosynthetic yield under low temperature stress. Our comprehensive multiomics analysis with transcription unit architecture provides foundational information on photosynthesis for future industrial strain development. IMPORTANCE Cyanobacteria are a compelling biochemical production platform for their ability to propagate using light and atmospheric CO2 via photosynthesis. However, the engineering of strains is hampered by limited understanding of photosynthesis under diverse environmental conditions such as high-light and low-temperature stresses. Herein, we decipher the transcriptomic and translatomic responses of the photosynthetic efficiency to stress conditions using the integrative analysis of multiomic data generated by RNA-seq and ribosome profiling, respectively. Through the generated massive data, along with the guide of the genome-wide transcription unit architecture constructed by transcription start sites and transcript 3′-end positions, we identified the factors affecting photosynthesis at transcription, posttranscription, and translation levels. Importantly, the high-light stress induces photodamage responses, and the low-temperature stress cripples the translation efficiency of photosynthesis-associated genes. The resulting insights provide pivotal information for future cyanobacterial cell factories powered by the engineering toward robust photosynthesis ability.

In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.