Translatomics in Action: Respiratory Illnesses
Respiratory illnesses encompass a wide range of disorders affecting the lungs and other parts of the respiratory system. These illnesses can vary from acute conditions like the common cold to chronic diseases such as asthma, lung cancer and as seen in recent times, SARS-CoV-2. The impact of respiratory illnesses on global health is profound, with millions of individuals affected annually, leading to significant morbidity, mortality, and economic burden (1).
Translatomics, utilizing techniques like ribosome profiling, offers precise insights into the translational control of gene expression, identifying specific proteins actively synthesized during respiratory illnesses (2). This technique reveals pathogen-host interactions and cellular responses at the translational level, aiding in the discovery of novel therapeutic targets and biomarkers, which could encourage the advancement of therapies and treatments for the many forms of respiratory illnesses that exist (3).
Comparative Analysis of Gene Expression in Virulent and Attenuated Strains of Infectious Bronchitis Virus at Subcodon Resolution
Journal of Virology, 2019
Dinan, A.M., Keep, S., Bickerton, S., Britton, P., Firth, A.E. and Brierley, I.
Backgrounds
Avian infectious bronchitis virus (IBV) is a major avian pathogen that significantly impacts the poultry industry by causing respiratory disease in chickens. To develop effective vaccination strategies, a comprehensive understanding of the molecular biology of IBV is urgently required. IBV, like other coronaviruses, has a long, single-stranded, positive-sense RNA genome approximately 27 kilobases (kb) in length and replicates by producing a nested set of subgenomic mRNAs (sgmRNAs). This study by Dinan et al. 2019 utilized advanced techniques—whole-transcriptome sequencing (RNA-seq) and ribosome profiling (Ribo-seq)—to explore the gene expression of IBV at subcodon resolution. They focused on two strains of the virus: the pathogenic M41-CK strain and the attenuated Beau-R strain. Study of both pathogenic and attenuated strains provided an insight into the translation and transcription of infected cells. This enabled a deeper understanding of gammcoronaviral gene expression and the variation that exists between species.
Key Findings
- Ribo-seq and RNA-seq analysis of cells infected with Beau-R and M41-CK strains of IBV, as well as mock-infected cells, revealed that there was a higher density of ribosome-protected fragments (RPFs) toward the 3′ ends of the viral genome, suggesting an active production of subgenomic mRNAs (sgmRNAs). In addition, a high positive-to-negative strand RNA ratio was observed, indicating active viral RNA synthesis and packaging over translation.
- Ribo-seq at the ORF1a/ORF1b junction showed efficient -1 programmed ribosomal frameshifting (-1 PRF), with 33% and 39% efficiency in Beau-R and M41-CK strains, respectively, consistent with other coronaviruses.
- Substantial ribosomal occupancy of two AUG-initiated ORFs, ORF4b and ORF4c, was confirmed, highlighting their translation in the viral genome.
- Analysis of translational efficiency (TE) revealed ORF4b is more efficiently translated than ORF4c, while the nucleocapsid (N) protein, despite its abundance, showed lower TE than other structural proteins.
- Several ribosomal pausing sites during IBV genome translation were identified, suggesting potential regulatory roles.
- Differential host gene expression analysis showed broad transcriptional and translational response similarities between Beau-R and M41-CK strains, with enriched immune-related pathways and significant differences in signalling receptor activity and heat shock protein-encoding genes between the strains.
Implications
Using RNA-seq and Ribo-seq, the study of IBV M41-CK and Beau-R strains revealed significant findings: two novel transcription junction sites, high-efficiency programmed ribosomal frameshifting (33-40%), and ribosomal pausing sites. These discoveries are crucial for advancing knowledge in the field of IBV because they offer detailed insights into viral RNA synthesis, gene expression, and translational control mechanisms. This enhanced understanding can lead to the identification of novel therapeutic targets and strategies to combat IBV, significantly improving our ability to manage and treat infections caused by this virus.
PM2. 5 promotes NSCLC carcinogenesis through translationally and transcriptionally activating DLAT-mediated glycolysis reprograming
Journal of Experimental & Clinical Cancer Research, 2022
Chen, Q., Wang, Y., Yang, L., Sun, L., Wen, Y., Huang, Y., Gao, K., Yang, W., Bai, F., Ling, L. and Zhou, Z.
Backgrounds
This study investigates the molecular mechanisms by which PM2.5, a significant risk factor for lung cancer in never-smokers, promotes non-small cell lung cancer (NSCLC). Traditional approaches have focused on mRNA expression, but they often overlook the complexities of protein synthesis and translation. By utilizing Ribo-seq and conventional RNA-seq, the researchers aimed to explore how PM2.5 affects gene translation, particularly in the glycolysis pathway. The study seeks to uncover how translation efficiency impacts on the activity of oncogenes and tumour suppressor genes in NSCLC, providing deeper insights into the translational landscape influenced by PM2.5 exposure and potentially revealing novel pathways involved in NSCLC development.
Key Findings
- It was observed that PM2.5 exposure induces substantial alterations in gene expression at both transcriptional and translational levels in human bronchial epithelial cells. Researchers identified 405 genes differentially expressed transcriptionally and 3501 genes translationally, with 3697 genes exhibiting changes in translation efficiency (TE), implicating that PM2.5 primarily influences cellular responses through translational mechanisms.
- PM2.5 exposure leads to significant TE shifts, particularly towards the glycolysis pathway in human bronchial epithelial cells. Pathway analysis showed that genes with altered TE were predominantly enriched in glycolysis, with DLAT emerging as the most significantly regulated gene, suggesting a critical role of translational alterations in the glycolysis pathway for PM2.5-induced pathogenesis.
- Significant increased expression of the glycolytic gene DLAT was observed when exposed to PM2.5 both in vitro and in vivo. These results indicate that DLAT is a primary target gene affected by PM2.5 exposure.
- DLAT overexpression in NSCLC cells increases glycolysis metabolism, indicated by higher extracellular levels of L-lactate and pyruvate. Conversely, DLAT knockdown reduced these glycolytic markers, suggesting that PM2.5 accelerates glycolysis in NSCLC cells by upregulating DLAT expression.
- It was found that DLAT promotes the malignancy of NSCLC cells by increasing cell viability and reducing apoptosis. Overexpression of DLAT in NSCLC cells led to significantly higher cell proliferation and decreased rates of apoptosis, while DLAT knockdown had the opposite effects, implying DLAT functions as an oncogene in NSCLC.
- PM2.5 exposure induced upregulation of eIF4E, a translation initiation factor that enhances DLAT translation. Experimental evidence shows that PM2.5 significantly increases eIF4E expression, leading to elevated DLAT levels in polysome fractions in cells overexpressing eIF4E and decreased levels in cells with eIF4E knockdown.
Implications
This study presents vital evidence that PM2.5-induced glycolysis is pivotal in NSCLC development, representing a significant stride in understanding environmental carcinogenesis. Employing translatomics, substantial PM2.5-induced alterations in gene expression were observed at both transcriptional and translational levels in human bronchial epithelial cells, emphasizing a predominant influence via translational mechanisms and notable shifts towards glycolysis. Moreover, by identifying DLAT as a key glycolytic gene and a novel oncogene in NSCLC, this research underscores DLAT’s dual role in promoting glycolysis and tumour progression. These findings not only enhance our understanding of the molecular mechanisms underlying PM2.5-induced cancer, but also propose DLAT as a potential therapeutic target for NSCLC treatment strategies.
SARS-CoV-2 infection engenders heterogeneous ribonucleoprotein interactions to impede translation elongation in the lungs
Experimental & Molecular Medicine, 2023
Kim, J., Youn, D., Choi, S., Lee, Y.W., Sumberzul, D., Yoon, J., Lee, H., Bae, J.W., Noh, H., On, D. and Hong, S.M.
Backgrounds
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in a global pandemic with millions of infections and fatalities. Despite vaccination efforts, effective treatments for COVID-19 remain limited, making a deeper understanding of viral behaviour and host responses during infection a necessity. Transcriptome studies have illuminated gene regulation during SARS-CoV-2 infection, yet mRNA changes do not always correlate with protein levels due to translational regulation complexities. Viruses, including SARS-CoV-2, can disrupt protein synthesis, influencing host responses crucial for disease progression. Ribosome profiling (Ribo-seq) offers a powerful tool to investigate these dynamics, capturing ribosome-RNA interactions and quantifying translation activity across the genome. Previous ribo-seq studies in cell lines have revealed insights into viral protein production and host response modulation. However, to comprehend SARS-CoV-2 pathophysiology fully, in vivo studies are essential to simulate tissue environments and diverse cell interactions observed in human patients and animal models. To address these gaps, this study applied tissue-optimized ribo-seq to severe-COVID-19 model mice to unveil temporal profiles of gene translation during infection. The aim was to identify unique translational signatures and molecular pathways that drive tissue pathology and immune responses, potentially revealing novel therapeutic targets for mitigating COVID-19 severity.
Key Findings
- SARS-CoV-2 shows distinct gene expression in mouse lung tissues versus cell lines, indicating significant virus-host interaction differences. Early infection increases viral genome presence, while later phases restrict viral proliferation and subgenomic RNA expression. Principal component analysis reveals differential viral gene expression between tissues and cell lines, influenced by tissue-specific interactions.
- During pathogenesis, pseudoribosomal ribonucleoprotein (RNP) interactions progressively develop in infected mouse lung tissues, resulting in compromised frame periodicities and increased untranslated region (UTR) enrichment in host genes. These noncanonical Ribo-seq read peaks and short read accumulations were more prevalent in tissues than in cell lines, indicating pseudoribosomal RNP interactions are specific to the tissue environment.
- It was found that during SARS-CoV-2 infection, ribosomes preferentially stall on transmembrane protein-coding mRNAs. This ribosome stalling is associated with attenuated protein synthesis and is particularly pronounced in ribosomal and focal adhesion-associated genes in the late phase of infection.
- Ribosome heterogeneity with compromised 5S ribonucleoprotein (rRNP) association emerges during pathogenesis, altering ribosomal protein interactions and impairing translation elongation. This is marked by decreased 5S rRNP-associated proteins, reduced p53 expression, and upregulation of cell cycle and proliferation-related genes, indicating a shift in ribosomal composition that promotes cell survival and proliferation.
- Pseudoribosomal RNP interactions in SARS-CoV-2 infected tissues result in widespread translational elongation impediments. Genes associated with these interactions show high nonsense-mediated decay sensitivity, intron retention, and are often related to microtubule functions. The translational suppression of these genes implies that pseudoribosomal RNPs hinder ribosome activity, especially in transcripts with short open reading frames.
Implications
The study found distinct gene expression profiles in SARS-CoV-2-infected lung tissues versus cell lines, highlighting significant virus-host interaction differences. Key findings include lower levels of subgenomic RNAs, pseudoribosomal RNP interactions, ribosome stalling on transmembrane protein-coding mRNAs, and ribosome heterogeneity impairing translation elongation. These insights reveal the complexity of viral pathogenesis in tissues, advancing understanding of SARS-CoV-2 mechanisms and informing potential therapeutic strategies.
References
1. Ekezie W, Jenkins AR, Hall IP, Evans C, Koju R, Kurmi OP, Bolton CE. The burden of chronic respiratory diseases in adults in Nepal: A systematic review. Chronic Respiratory Disease. 2021 Jun 30;18:1479973121994572.
2. Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. science. 2009 Apr 10;324(5924):218-23.
3. Wang S, Huang T, Xie Z, Wan L, Ren H, Wu T, Xie L, Luo S, Li M, Xie Z, Fan Q. Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus. Viruses. 2023 Nov 29;15(12):2346.
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