Effects of SARS-CoV-2 Membrane Glycoprotein on Host Pre-mRNA 3' Processing

Objective To investigate the effect of SARS-CoV-2 membrane protein on the processing of the 3' untranslated region (UTR) of the mRNA precursor (pre-mRNA) in host cells. Methods Based on the cell model of human lung epithelial cells A549, over-expression of the SARS-CoV-2 membrane protein was performed. The RNA-Seq high-throughput sequencing technique and bioinformatics methods was employed to analyze the systematic characterization of alternative polyadenylation (APA) events in host cells. Genes with significant APA events were uploaded to the Metascape database for functional enrichment analysis. In addition, alternative 3'UTR length of genes with APA events was verified by RT-qPCR. Then the target protein expression level was detected by Western blot. Results A total of 813 genes that were significant dynamic APA events in host cells that over-expressed SARS-CoV-2 membrane protein. These genes were enriched in cell biologicial processes such as the mitotic cell cycle and regulation of cellular response to stress. We further screened AKT1, which encodes a critical regulator involved in the above biological process, showing a 3'UTR lengthening in IGV software. RT-qPCR verified the trend of 3'UTR length changes of AKT1. Western blot showed the increased level of phosphorylated AKT1 protein in over-expressed group of M protein. Conclusion SARS-CoV-2 membrane protein potentially affects the 3' processing of host pre-mRNAs. AKT1, which is involved in a variety of viral biological processes, with 3'UTR lengthening, and its protein function was activated intracellularly. © 2022 Institute of Biophysics,Chinese Academy of Sciences. All rights reserved.

Post-transcriptional control of haemostatic genes: mechanisms and emerging therapeutic concepts in thrombo-inflammatory disorders.

The haemostatic system is pivotal to maintaining vascular integrity. Multiple components involved in blood coagulation have central functions in inflammation and immunity. A derailed haemostasis is common in prevalent pathologies such as sepsis, cardiovascular disorders, and lately, COVID-19. Physiological mechanisms limit the deleterious consequences of a hyperactivated haemostatic system through adaptive changes in gene expression. While this is mainly regulated at the level of transcription, co- and posttranscriptional mechanisms are increasingly perceived as central hubs governing multiple facets of the haemostatic system. This layer of regulation modulates the biogenesis of haemostatic components, for example in situations of increased turnover and demand. However, they can also be 'hijacked' in disease processes, thereby perpetuating and even causally entertaining associated pathologies. This review summarizes examples and emerging concepts that illustrate the importance of posttranscriptional mechanisms in haemostatic control and crosstalk with the immune system. It also discusses how such regulatory principles can be used to usher in new therapeutic concepts to combat global medical threats such as sepsis or cardiovascular disorders.

Effects of SARS-CoV-2 Membrane Glycoprotein on Host Pre-mRNA 3 ' Processing

Objective To investigate the effect of SARS-CoV-2 membrane protein on the processing of the 3' untranslated region (UTR) of the mRNA precursor (pre-mRNA) in host cells. Methods Based on the cell model of human lung epithelial cells A549, over-expression of the SARS-CoV-2 membrane protein was performed. The RNA-Seq high-throughput sequencing technique and bioinformatics methods was employed to analyze the systematic characterization of alternative polyadenylation (APA) events in host cells. Genes with significant APA events were uploaded to the Metascape database for functional enrichment analysis. In addition, alternative 3'UTR length of genes with APA events was verified by RT-qPCR. Then the target protein expression level was detected by Western blot. Results A total of 813 genes that were significant dynamic APA events in host cells that overexpressed SARS-CoV-2 membrane protein. These genes were enriched in cell biologicial processes such as the mitotic cell cycle and regulation of cellular response to stress. We further screened AKT1, which encodes a critical regulator involved in the above biological process, showing a 3'UTR lengthening in IGV software. RT-qPCR verified the trend of 3'UTR length changes of AKT1. Western blot showed the increased level of phosphorylated AKT1 protein in over-expressed group of M protein. Conclusion SARS-CoV-2 membrane protein potentially affects the 3' processing of host pre-mRNAs. AKT1, which is involved in a variety of viral biological processes, with 3'UTR lengthening, and its protein function was activated intracellularly.

Multifaceted Applications of Genetically Modified Micro-organisms: A Biotechnological Revolution.

BACKGROUND: Genetically modified micro-organisms like bacteria, viruses, algae and fungi are novel approaches used in the field of healthcare due to better efficacy and targeted delivery in comparison to conventional approaches. OBJECTIVES: This review article focuses on the applications of genetically modified micro-organisms in the treatment of cancer, obesity and HIV infection. The gut microbiome causes metabolic disorders, however, the use of genetically modified bacteria alters the gut microbiota and delivers therapeutically effective drugs in the treatment of obesity. METHODS: Enhancement of the therapeutic activity of different micro-organisms is required for multiple treatments in cancer, diabetes, etc., by incorporating their fragments into the microbial filaments with the help of genetic modification approaches. Various methods like amelioration of NAPE synthesis, silica immobilization, polyadenylation and electrochemical are used to integrate the strain into the bacteria and engineer a live virus with a peptide. RESULTS: The development of novel microbial strains using genetic modifications over core strains offers higher precision, greater molecular multiplicity, better prevention from the degradation of microbes in atmospheric temperature and significant reduction of side effects for therapeutic applications. Moreover, genetically modified micro-organisms are used in multidisciplinary sectors like generation of electricity, purification of water, bioremediation process, etc., indicating the versatility and scope of genetically engineered microbes. CONCLUSION: The bioengineered micro-organisms with genetic modifications proved to be advantageous in various conditions like cancer, diabetes, malaria, organ regeneration, inflammatory bowel disease, etc. This article provides insight into various applications of genetically modified microbes in different sectors with their implementation for regulatory approval.

Analysis of Polyadenylation Signal Usage with Full-Length Transcriptome in Spodoptera frugiperda (Lepidoptera: Noctuidae).

During the messenger RNA (mRNA) maturation process, RNA polyadenylation is a key step, and is coupled to the termination of transcription. Various cis-acting elements near the cleavage site and their binding factors would affect the process of polyadenylation, and AAUAAA, a highly conserved hexamer, was the most important polyadenylation signal (PAS). PAS usage is one of the critical modification determinants targeted at mRNA post-transcription. The full-length transcriptome has recently generated a massive amount of sequencing data, revealing poly(A) variation and alternative polyadenylation (APA) in Spodoptera frugiperda. We identified 50,616 polyadenylation signals in Spodoptera frugiperda via analysis of full-length transcriptome combined with expression Sequence Tags Technology (EST). The polyadenylation signal usage in Spodoptera frugiperda is conserved, and it is similar to that of flies and other animals. AAUAAA and AUUAAA are the most highly conserved polyadenylation signals of all polyadenylation signals we identified. Additionally, we found the U/GU-rich downstream sequence element (DSE) in the cleavage site. These results demonstrate that APA in Spodoptera frugiperda plays a significant role in root growth and development. This is the first polyadenylation signal usage analysis in agricultural pests, which can deepen our understanding of Spodoptera frugiperda and provide a theoretical basis for pest control.

mRNA Vaccine: Determinants of Clinical Efficacy, and Optimization of Pharmacological Effects

The messenger ribonucleic acid (mRNA) vaccine has proven to be beneficial in containing the severe acute respiratory syndrome coronavirus. However, some school thought that it is not as effective as proposed, even though many others attest to its efficacy. The study employed scoping review on online journal and book publications to reveal the pharmacokinetics and pharmacodynamics of the mRNA vaccine. In this work, we have discussed the mechanism of action of the self-amplified messenger ribonucleic acid vaccines, the determinants of the clinical efficacy, and the possible reasons behind the varying efficacies that different populations may experience. We provided possible ways to tackle this challenge. In providing these solutions, we discussed pharmacogenetics viz-a-viz epigenetics. The study also used figures to elaborate on the factors that determine the clinical efficacy of mRNA vaccines. We are confident that caregivers and public health officials in the tropics would find the information interesting and invaluable.

Long-Read RNA Sequencing Identifies Polyadenylation Elongation and Differential Transcript Usage of Host Transcripts During SARS-CoV-2 In Vitro Infection.

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

Genome-Wide Profiling Reveals Alternative Polyadenylation of Innate Immune-Related mRNA in Patients With COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused many deaths worldwide. To date, the mechanism of viral immune escape remains unclear, which is a great obstacle to developing effective clinical treatment. RNA processing mechanisms, including alternative polyadenylation (APA) and alternative splicing (AS), are crucial in the regulation of most human genes in many types of infectious diseases. Because the role of APA and AS in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown, we performed de novo identification of dynamic APA sites using a public dataset of human peripheral blood mononuclear cell (PBMC) RNA-Seq data in COVID-19 patients. We found that genes with APA were enriched in innate immunity -related gene ontology categories such as neutrophil activation, regulation of the MAPK cascade and cytokine production, response to interferon-gamma and the innate immune response. We also reported genome-wide AS events and enriched viral transcription-related categories upon SARS-CoV-2 infection. Interestingly, we found that APA events may give better predictions than AS in COVID-19 patients, suggesting that APA could act as a potential therapeutic target and novel biomarker in those patients. Our study is the first to annotate genes with APA and AS in COVID-19 patients and highlights the roles of APA variation in SARS-CoV-2 infection.

Computational Methods to Study Human Transcript Variants in COVID-19 Infected Lung Cancer Cells.

Microbes and viruses are known to alter host transcriptomes by means of infection. In light of recent challenges posed by the COVID-19 pandemic, a deeper understanding of the disease at the transcriptome level is needed. However, research about transcriptome reprogramming by post-transcriptional regulation is very limited. In this study, computational methods developed by our lab were applied to RNA-seq data to detect transcript variants (i.e., alternative splicing (AS) and alternative polyadenylation (APA) events). The RNA-seq data were obtained from a publicly available source, and they consist of mock-treated and SARS-CoV-2 infected (COVID-19) lung alveolar (A549) cells. Data analysis results show that more AS events are found in SARS-CoV-2 infected cells than in mock-treated cells, whereas fewer APA events are detected in SARS-CoV-2 infected cells. A combination of conventional differential gene expression analysis and transcript variants analysis revealed that most of the genes with transcript variants are not differentially expressed. This indicates that no strong correlation exists between differential gene expression and the AS/APA events in the mock-treated or SARS-CoV-2 infected samples. These genes with transcript variants can be applied as another layer of molecular signatures for COVID-19 studies. In addition, the transcript variants are enriched in important biological pathways that were not detected in the studies that only focused on differential gene expression analysis. Therefore, the pathways may lead to new molecular mechanisms of SARS-CoV-2 pathogenesis.

The interface between coronaviruses and host cell RNA biology: Novel potential insights for future therapeutic intervention.

Coronaviruses, including SARS-Cov-2, are RNA-based pathogens that interface with a large variety of RNA-related cellular processes during infection. These processes include capping, polyadenylation, localization, RNA stability, translation, and regulation by RNA binding proteins or noncoding RNA effectors. The goal of this article is to provide an in-depth perspective on the current state of knowledge of how various coronaviruses interact with, usurp, and/or avoid aspects of these cellular RNA biology machineries. A thorough understanding of how coronaviruses interact with RNA-related posttranscriptional processes in the cell should allow for new insights into aspects of viral pathogenesis as well as identify new potential avenues for the development of anti-coronaviral therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.