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Temperature
Alternative splicing
Innate immunity
SARS-CoV-2
Abstract:
Alternative splicing (AS) is a dynamic and highly regulated process responsible for increasing the number of unique proteins encoded by a single gene, generating proteomic diversity. Additionally, AS allows cells to quickly respond to internal and external changes, such as temperature variation. The body temperature in homeothermic organisms oscillates in a circadian manner in a range of around 1-4°C. These subtle changes in core body temperature are sufficient to impact AS and gene expression by altering SR protein phosphorylation. In addition to physiological changes in body temperature, pathological changes can also occur, for example, during infection with fever. Elevated core body temperature during infection is important for pathogens clearance and the activation of the host immune cells. However, little is known about the temperature-sensing machinery that triggers changes in immune cell behavior. In this thesis, we started addressing this by performing RNA-seq with a macrophage cell line (RAW 264.7) incubated at 34°C, 37°C, and 38°C for 12h. Notably, increasing the temperature from 37°C to 38°C upregulated the expression of antiviral genes, including members of the JAK/STAT pathway. These results suggested that individual and age-dependent body temperature variations can control the expression of the antiviral genes, with higher temperatures leading to higher expression. Furthermore, we observed increased Stat2 protein and phosphorylation at higher temperatures (39°C) in RAW 264.7 cells. Inhibition of the JAK/STAT pathway prevented the temperature-controlled increase of all antiviral genes tested and reduced their basal expression at 37°C. Moreover, we showed that elevated temperature strongly increases nitric oxide production, another branch of antiviral immunity. Furthermore, we identified a temperature-dependent alternative 5’ splice site in Stat2 exon 11. The usage of the alternative 5’ splice site was higher at lower temperatures (35°C and 37°C) and it leads to the inclusion of a premature termination codon (PTC), which will prevent the formation of a functional protein and lead to degradation via nonsense-mediated decay (NMD). By blocking the NMD pathway using cycloheximide (CHX), we observed an increased abundance of the PTC-containing isoform and total Stat2 mRNA in the CHX-treated cells. To further validate the influence of the alternative 5’ splice site of exon 11 in Stat2 on the expression of antiviral genes, we used antisense oligonucleotides targeting this splice site and a CRISPR/Cas9-edited cell line lacking the alternative 5’ splice site. Both approaches showed an increased expression of Stat2 and other interferon-stimulated genes (ISGs) in the cells with reduced or depleted usage of the alternative 5’ splice site. Additionally, SR proteins knockdown (KD) showed that Srsf1 regulates the usage of this splice site. Srsf1 KD decreased the usage of the alternative 5’ splice site and led to increased expression of Stat2 and other ISGs. Thus, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity. Finally, using a cell culture model we observed that higher temperature correlates with reduced SARS-CoV-2 replication. We then hypothesized that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. In conclusion, we showed that alternative splicing coupled with NMD decreases the expression of Stat2 at lower temperatures. The upregulation of Stat2 at higher temperatures in conjunction with JAK1 activity induces the expression of antiviral genes, which mounts a successful antiviral response, leading to lower viral replication.
