Competing endogenous RNAs in human astrocytes: crosstalk and interacting networks in response to lipotoxicity.

Neurodegenerative diseases (NDs) are characterized by a progressive deterioration of neuronal function, leading to motor and cognitive damage in patients. Astrocytes are essential for maintaining brain homeostasis, and their functional impairment is increasingly recognized as central to the etiology of various NDs. Such impairment can be induced by toxic insults with palmitic acid (PA), a common fatty acid, that disrupts autophagy, increases reactive oxygen species, and triggers inflammation. Although the effects of PA on astrocytes have been addressed, most aspects of the dynamics of this fatty acid remain unknown. Additionally, there is still no model that satisfactorily explains how astroglia goes from being neuroprotective to neurotoxic. Current incomplete knowledge needs to be improved by the growing field of non-coding RNAs (ncRNAs), which is proven to be related to NDs, where the complexity of the interactions among these molecules and how they control other RNA expressions need to be addressed. In the present study, we present an extensive competing endogenous RNA (ceRNA) network using transcriptomic data from normal human astrocyte (NHA) cells exposed to PA lipotoxic conditions and experimentally validated data on ncRNA interaction. The obtained network contains 7 lncRNA transcripts, 38 miRNAs, and 239 mRNAs that showed enrichment in ND-related processes, such as fatty acid metabolism and biosynthesis, FoxO and TGF-ß signaling pathways, prion diseases, apoptosis, and immune-related pathways. In addition, the transcriptomic profile was used to propose 22 potential key controllers lncRNA/miRNA/mRNA axes in ND mechanisms. The relevance of five of these axes was corroborated by the miRNA expression data obtained in other studies. MEG3 (ENST00000398461)/hsa-let-7d-5p/ATF6B axis showed importance in Parkinson's and late Alzheimer's diseases, while AC092687.3/hsa-let-7e-5p/[SREBF2, FNIP1, PMAIP1] and SDCBP2-AS1 (ENST00000446423)/hsa-miR-101-3p/MAPK6 axes are probably related to Alzheimer's disease development and pathology. The presented network and axes will help to understand the PA-induced mechanisms in astrocytes, leading to protection or injury in the CNS under lipotoxic conditions as part of the intricated cellular regulation influencing the pathology of different NDs. Furthermore, the five corroborated axes could be considered study targets for new pharmacologic treatments or as possible diagnostic molecules, contributing to improving the quality of life of millions worldwide.

Computational Prediction of RNA-RNA Interactions between Small RNA Tracks from Betacoronavirus Nonstructural Protein 3 and Neurotrophin Genes during Infection of an Epithelial Lung Cancer Cell Line: Potential Role of Novel Small Regulatory RNA.

Whether RNA-RNA interactions of cytoplasmic RNA viruses, such as Betacoronavirus, might end in the biogenesis of putative virus-derived small RNAs as miRNA-like molecules has been controversial. Even more, whether RNA-RNA interactions of wild animal viruses may act as virus-derived small RNAs is unknown. Here, we address these issues in four ways. First, we use conserved RNA structures undergoing negative selection in the genomes of SARS-CoV, MERS-CoV, and SARS-CoV-2 circulating in different bat species, intermediate animals, and human hosts. Second, a systematic literature review was conducted to identify Betacoronavirus-targeting hsa-miRNAs involved in lung cell infection. Third, we employed sophisticated long-range RNA-RNA interactions to refine the seed sequence homology of hsa-miRNAs with conserved RNA structures. Fourth, we used high-throughput RNA sequencing of a Betacoronavirus-infected epithelial lung cancer cell line (Calu-3) to validate the results. We proposed nine potential virus-derived small RNAs: two vsRNAs in SARS-CoV (Bats: SB-vsRNA-ORF1a-3p; SB-vsRNA-S-5p), one vsRNA in MERS-CoV (Bats: MB-vsRNA-ORF1b-3p), and six vsRNAs in SARS-CoV-2 (Bats: S2B-vsRNA-ORF1a-5p; intermediate animals: S2I-vsRNA-ORF1a-5p; and humans: S2H-vsRNA-ORF1a-5p, S2H-vsRNA-ORF1a-3p, S2H-vsRNA-ORF1b-3p, S2H-vsRNA-ORF3a-3p), mainly encoded by nonstructural protein 3. Notably, Betacoronavirus-derived small RNAs targeted 74 differentially expressed genes in infected human cells, of which 55 upregulate the molecular mechanisms underlying acute respiratory distress syndrome (ARDS), and the 19 downregulated genes might be implicated in neurotrophin signaling impairment. These results reveal a novel small RNA-based regulatory mechanism involved in neuropathogenesis that must be further studied to validate its therapeutic use.

Palmitic Acid Upregulates Type I Interferon-Mediated Antiviral Response and Cholesterol Biosynthesis in Human Astrocytes.

Chronic intake of a high-fat diet increases saturated fatty acids in the brain causing the progression of neurodegenerative diseases. Palmitic acid is a free fatty acid abundant in the diet that at high concentrations may penetrate the blood-brain barrier and stimulate the production of pro-inflammatory cytokines, leading to inflammation in astrocytes. The use of the synthetic neurosteroid tibolone in protection against fatty acid toxicity is emerging, but its transcriptional effects on palmitic acid-induced lipotoxicity remain unclear. Herein, we performed a transcriptome profiling of normal human astrocytes to investigate the molecular mechanisms by which palmitic acid causes cellular damage to astrocytes, and whether tibolone could reverse its detrimental effects. Astrocytes undergo a profound transcriptional change at 2 mM palmitic acid, affecting the expression of 739 genes, 366 upregulated and 373 downregulated. However, tibolone at 10 nM does not entirely reverse palmitic acid effects. Additionally, the protein-protein interaction reveals two novel gene clustering modules. The first module involves astrocyte defense responses by upregulation of pathways associated with antiviral innate immunity, and the second is linked to lipid metabolism. Our data suggest that activation of viral response signaling pathways might be so far, the initial molecular mechanism of astrocytes in response to a lipotoxic insult by palmitic acid, triggered particularly upon increased expression levels of IFIT2, IRF1, and XAF1. Therefore, this novel approach using a global gene expression analysis may shed light on the pleiotropic effects of palmitic acid on astrocytes, and provide a basis for future studies addressed to elucidate these responses in neurodegenerative conditions, which is highly valuable for the design of therapeutic strategies.

RNA structure-altering mutations underlying positive selection on Spike protein reveal novel putative signatures to trace crossing host-species barriers in Betacoronavirus.

Similar to other RNA viruses, the emergence of Betacoronavirus relies on cross-species viral transmission, which requires careful health surveillance monitoring of protein-coding information as well as genome-wide analysis. Although the evolutionary jump from natural reservoirs to humans may be mainly traced-back by studying the effect that hotspot mutations have on viral proteins, it is largely unexplored if other impacts might emerge on the structured RNA genome of Betacoronavirus. In this survey, the protein-coding and viral genome architecture were simultaneously studied to uncover novel insights into cross-species horizontal transmission events. We analysed 1,252,952 viral genomes of SARS-CoV, MERS-CoV, and SARS-CoV-2 distributed across the world in bats, intermediate animals, and humans to build a new landscape of changes in the RNA viral genome. Phylogenetic analyses suggest that bat viruses are the most closely related to the time of most recent common ancestor of Betacoronavirus, and missense mutations in viral proteins, mainly in the S protein S1 subunit: SARS-CoV (G > T; A577S); MERS-CoV (C > T; S746R and C > T; N762A); and SARS-CoV-2 (A > G; D614G) appear to have driven viral diversification. We also found that codon sites under positive selection on S protein overlap with non-compensatory mutations that disrupt secondary RNA structures in the RNA genome complement. These findings provide pivotal factors that might be underlying the eventual jumping the species barrier from bats to intermediate hosts. Lastly, we discovered that nearly half of the Betacoronavirus genomes carry highly conserved RNA structures, and more than 90% of these RNA structures show negative selection signals, suggesting essential functions in the biology of Betacoronavirus that have not been investigated to date. Further research is needed on negatively selected RNA structures to scan for emerging functions like the potential of coding virus-derived small RNAs and to develop new candidate antiviral therapeutic strategies.

Phylogenetic analysis of Alphapapillomavirus based on L1, E6 and E7 regions suggests that carcinogenicity and tissue tropism have appeared multiple times during viral evolution.

Members of the Alphapapillomavirus genus are causative agents for cervix cancer and benign lesions in humans. These viruses are classified according to sequence similarities in their L1 region. Yet, viral carcinogenicity has been associated with variations in the proteins encoded by the E6 and E7 genes. In order to relate evolutionary history with origin of carcinogenicity, we performed phylogenetic reconstructions using both nucleotide and predicted amino acid sequences of the L1, E6 and E7 genes. Whilst phylogenetic analysis of L1 reconstructed genus evolutionary history, phylogenies based on E6 and E7 proteins support the idea that mutations at amino acids S/Tx [V/L] (E6) and LxCxE (E7) might be responsible for carcinogenic potential. These findings indicate that virulence within Alphapapillomavirus have appeared multiple times during evolution. Our results reveal that oncogenic potential is not a monophyletic clade-specific adaptation but might be the result of positive selection on random mutations occurring on proteins involved in host infection during viral diversification.