Search Results (33)

Gene expression is a tightly regulated process that involves multiple layers of control, including transcriptional, post-transcriptional, and translational regulation. To gain a comprehensive understanding of gene expression dynamics and its functional implications, it is crucial to compare translatomic, transcriptomic, and proteomic data. The two most common analysis methods, Ribo-seq and RNC-Seq, were used to analyze the translatome of the same sample, whose datasets were downloaded from the TranslatomeDB database. The resulting translatome maps obtained for three cell lines (HBE, A549, and MCF-7) using these two methods were comparatively analyzed. The two methods of translatome analysis were shown to provide comparable results and can be used interchangeably. The obtained mRNA translation patterns were annotated in the transcriptome and proteome context for the same sample, which may become the basis for the reconstruction of the molecular mechanisms of pathological process development in the future. Full article

Polyploidy, a prevalent event in plant evolution, drives phenotypic diversification and speciation. While transcriptional changes and regulation in polyploids have been extensively studied, the translational level impact remains largely unexplored. To address this gap, we conducted a comparative transcriptomic and translatomic analysis of cotton leaves from allopolyploid species G. hirsutum (AD₁) and G. barbadense (AD₂) relative to their model A-genome and D-genome diploid progenitors. Our data revealed that while allopolyploidization significantly affects the transcriptional landscape, its impact on translation was relatively modest, evidenced by a narrower expression range and fewer expression changes in ribosome-protected fragments than in mRNA levels. Allopolyploid-specific changes commonly identified in both AD₁ and AD₂ were observed in 7393 genes at either transcriptional or translational levels. Interestingly, the majority of translational changes exhibited concordant down-regulation in both ribosome-protected fragments and mRNA, particularly associated with terpenoid synthesis and metabolism (352 genes). Regarding translational efficiency (TE), at least one-fifth of cotton genes exhibit translational level regulation, with a general trend of more down-regulation (13.9–15.1%) than up-regulation (7.3–11.2%) of TE. The magnitude of translational regulation was slightly reduced in allopolyploids compared with diploids, and allopolyploidy tends to have a more profound impact on genes and functional associations with ultra-low TE. Moreover, we demonstrated a reduced extent of homeolog expression biases during translation compared with transcription. Our study provides insights into the regulatory consequences of allopolyploidy post-transcription, contributing to a comprehensive understanding of regulatory mechanisms of duplicated gene expression evolution. Full article

Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) is an important economic cold-water fish that is susceptible to heat stress. To date, the heat stress response in rainbow trout is more widely understood at the transcriptional level, while little research has been conducted at the translational level. To reveal the translational regulation of heat stress in rainbow trout, in this study, we performed a ribosome profiling assay of rainbow trout liver under normal and heat stress conditions. Comparative analysis of the RNA-seq data with the ribosome profiling data showed that the folding changes in gene expression at the transcriptional level are moderately correlated with those at the translational level. In total, 1213 genes were significantly altered at the translational level. However, only 32.8% of the genes were common between both levels, demonstrating that heat stress is coordinated across both transcriptional and translational levels. Moreover, 809 genes exhibited significant differences in translational efficiency (TE), with the TE of these genes being considerably affected by factors such as the GC content, coding sequence length, and upstream open reading frame (uORF) presence. In addition, 3468 potential uORFs in 2676 genes were identified, which can potentially affect the TE of the main open reading frames. In this study, Ribo-seq and RNA-seq were used for the first time to elucidate the coordinated regulation of transcription and translation in rainbow trout under heat stress. These findings are expected to contribute novel data and theoretical insights to the international literature on the thermal stress response in fish. Full article

Small open reading frames (sORFs; <300 nucleotides or <100 amino acids) are widespread across all genomes, and an increasing variety of them appear to be translating from non-genic regions. Over the past few decades, peptides produced from sORFs have been identified as functional in various organisms, from bacteria to humans. Despite recent advances in next-generation sequencing and proteomics, accurate annotation and classification of sORFs remain a rate-limiting step toward reliable and high-throughput detection of small proteins from non-genic regions. Additionally, the cost of computational methods utilizing machine learning is lower than that of biological experiments, and they can be employed to detect sORFs, laying the groundwork for biological experiments. We present D-sORF, a machine-learning framework that integrates the statistical nucleotide context and motif information around the start codon to predict coding sORFs. D-sORF scores directly for coding identity and requires only the underlying genomic sequence, without incorporating parameters such as the conservation, which, in the case of sORFs, may increase the dispersion of scores within the significantly less conserved non-genic regions. D-sORF achieves 94.74% precision and 92.37% accuracy for small ORFs (using the 99 nt medium length window). When D-sORF is applied to sORFs associated with ribosomes, the identification of transcripts producing peptides (annotated by the Ensembl IDs) is similar to or superior to experimental methodologies based on ribosome-sequencing (Ribo-Seq) profiling. In parallel, the recognition of putative negative data, such as the intron-containing transcripts that associate with ribosomes, remains remarkably low, indicating that D-sORF could be efficiently applied to filter out false-positive sORFs from Ribo-Seq data because of the non-productive ribosomal binding or noise inherent in these protocols. Full article

Regulation of translation is a crucial step in gene expression. Developmental signals and environmental stimuli dynamically regulate translation via upstream small open reading frames (uORFs) and ribosome pausing. Recent studies have revealed many plant genes that are specifically regulated by uORF translation following changes in growth conditions, but ribosome-pausing events are less well understood. In this study, we performed ribosome profiling (Ribo-seq) of etiolated maize (Zea mays) seedlings exposed to light for different durations, revealing hundreds of genes specifically regulated at the translation level during the early period of light exposure. We identified over 400 ribosome-pausing events in the dark that were rapidly released after illumination. These results suggested that ribosome pausing negatively regulates translation from specific genes, a conclusion that was supported by a non-targeted proteomics analysis. Importantly, we identified a conserved nucleotide motif downstream of the pausing sites. Our results elucidate the role of ribosome pausing in the control of gene expression in plants; the identification of the cis-element at the pausing sites provides insight into the mechanisms behind translation regulation and potential targets for artificial control of plant translation. Full article

Nitrogen (N) availability determines higher plant productivity and yield. However, the molecular mechanisms governing N acquisition and utilization remain largely unknown in maize. In this study, ATAC-seq, RNA-seq, and Ribo-seq analyses were conducted in maize roots under different N supply conditions. A set of differentially expressed genes enriched in N and phenylpropanoid metabolisms at both the transcription and translation levels were highlighted. Interestingly, less than half of low-N responsive genes were shared between transcription and translation. The alteration of translational efficiency (TE) is also an important mechanism by which maize responds to LN. In addition, we identified low-N-induced open chromatin regions (OCRs) and observed an enrichment of transcription factor (TF) binding motifs. Furthermore, we constructed a transcriptional regulatory network for maize roots subjected to low-N. These findings extend our understanding of N availability response and provide new insights for improving N use efficiency (NUE). Full article

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-β2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-β2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome. Full article

Y-box-binding proteins (YB proteins) are multifunctional DNA- and RNA-binding proteins that play an important role in the regulation of gene expression. The high homology of their cold shock domains and the similarity between their long, unstructured C-terminal domains suggest that Y-box-binding proteins may have similar functions in a cell. Here, we consider the functional interchangeability of the somatic YB proteins YB-1 and YB-3. RNA-seq and Ribo-seq are used to track changes in the mRNA abundance or mRNA translation in HEK293T cells solely expressing YB-1, YB-3, or neither of them. We show that YB proteins have a dual effect on translation. Although the expression of YB proteins stimulates global translation, YB-1 and YB-3 inhibit the translation of their direct CLIP-identified mRNA targets. The impact of YB-1 and YB-3 on the translation of their mRNA targets is similar, which suggests that they can substitute each other in inhibiting the translation of their mRNA targets in HEK293T cells. Full article

Escherichia coli is a benchmark organism, which has been deeply studied by the scientific community for decades, obtaining a vast amount of metabolic and genetic data. Among these data, estimates of the translation speed of ribosomes over their genome are available. These estimates are based on Ribo-Seq profiles, where the abundance of a particular fragment of mRNA in a profile indicates that it was sampled many times inside a cell. Various measurements of Ribo-Seq profiles are available for Escherichia coli, yet they do not always show a high degree of correspondence, which means that they can vary significantly in different experimental setups, being characterized by poor reproducibility. Indeed, within Ribo-Seq profiles, the translation speed for some sequences is easier to estimate, while for others, an uneven distribution of consensus among the different estimates is evidenced. Our goal is to develop an artificial intelligence method that can be trained on a small pool of highly reproducible sequences to establish their translation rate, which can then be exploited to calculate a more reliable estimate of the translation speed on the rest of the genome. Full article

Avian reovirus (ARV) infection is prevalent in farmed poultry and causes viral arthritis and severe immunosuppression. The spleen plays a very important part in protecting hosts against infectious pathogens. In this research, transcriptome and translatome sequencing technology were combined to investigate the mechanisms of transcriptional and translational regulation in the spleen after ARV infection. On a genome-wide scale, ARV infection can significantly reduce the translation efficiency (TE) of splenic genes. Differentially expressed translational efficiency genes (DTEGs) were identified, including 15 upregulated DTEGs and 396 downregulated DTEGs. These DTEGs were mainly enriched in immune regulation signaling pathways, which indicates that ARV infection reduces the innate immune response in the spleen. In addition, combined analyses revealed that the innate immune response involves the effects of transcriptional and translational regulation. Moreover, we discovered the key gene IL4I1, the most significantly upregulated gene at both the transcriptional and translational levels. Further studies in DF1 cells showed that overexpression of IL4I1 could inhibit the replication of ARV, while inhibiting the expression of endogenous IL4I1 with siRNA promoted the replication of ARV. Overexpression of IL4I1 significantly downregulated the mRNA expression of IFN-β, LGP2, TBK1 and NF-κB; however, the expression of these genes was significantly upregulated after inhibition of IL4I1, suggesting that IL4I1 may be a negative feedback effect of innate immune signaling pathways. In addition, there may be an interaction between IL4I1 and ARV σA protein, and we speculate that the IL4I1 protein plays a regulatory role by interacting with the σA protein. This study not only provides a new perspective on the regulatory mechanisms of the innate immune response after ARV infection but also enriches the knowledge of the host defense mechanisms against ARV invasion and the outcome of ARV evasion of the host’s innate immune response. Full article

Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses (e.g., transcriptomics, proteomics, and phylogenetics). In this study, we improved the annotation of the Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their potential role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies. Full article

CircRNAs are newly identified special endogenous RNA molecules that covalently close a loop by back-splicing with pre-mRNA. In the cytoplasm, circRNAs would act as molecular sponges to bind with specific miRNA to promote the expression of target genes. However, knowledge of circRNA functional alternation in skeletal myogenesis is still in its infancy. In this study, we identified a circRNA–miRNA–mRNA interaction network in which the axis may be implicated in the progression of chicken primary myoblasts’ (CPMs) myogenesis by multi-omics (i.e., circRNA-seq and ribo-seq). In total, 314 circRNA–miRNA–mRNA regulatory axes containing 66 circRNAs, 70 miRNAs, and 24 mRNAs that may be relevant to myogenesis were collected. With these, the circPLXNA2-gga-miR-12207-5P-MDM4 axis aroused our research interest. The circPLXNA2 is highly differentially expressed during differentiation versus proliferation. It was demonstrated that circPLXNA2 inhibited the process of apoptosis while at the same time stimulating cell proliferation. Furthermore, we demonstrated that circPLXNA2 could inhibit the repression of gga-miR-12207-5p to MDM4 by directing binding to gga-miR-12207-5p, thereby restoring MDM4 expression. In conclusion, circPLXNA2 could function as a competing endogenous RNA (ceRNA) to recover the function of MDM4 by directing binding to gga-miR-12207-5p, thereby regulating the myogenesis. Full article

Protein synthesis is tightly regulated by both gene-specific and global mechanisms to match the metabolic and proliferative demands of the cell. While the regulation of global protein synthesis in response to mitogen or stress signals is relatively well understood in multiple experimental systems, how different cell types fine-tune their basal protein synthesis rate is not known. In a previous study, we showed that resting B and T lymphocytes exhibit dramatic differences in their metabolic profile, with implications for their post-activation function. Here, we show that resting B cells, despite being quiescent, exhibit increased protein synthesis in vivo as well as ex vivo. The increased protein synthesis in B cells is driven by mTORC1, which exhibits an intermediate level of activation in these cells when compared with resting T cells and activated B cells. A comparative analysis of the transcriptome and translatome of these cells indicates that the genes encoding the MHC Class II molecules and their chaperone CD74 are highly translated in B cells. These data suggest that the translatome of B cells shows enrichment for genes associated with antigen processing and presentation. Even though the B cells exhibit higher mTORC1 levels, they prevent the translational activation of TOP mRNAs, which are mostly constituted by ribosomal proteins and other translation factors, by upregulating 4EBP1 levels. This mechanism may keep the protein synthesis machinery under check while enabling higher levels of translation in B cells. Full article

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. Full article

Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 (SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNA^Sec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3′ of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2^R543Q-mutant brains. Full article