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We read with great interest the study by Salto-Alejandre et al. showing that SARS-CoV-2 viral load and interferon (IFN)-γ-mediated immunity, crucial in the acute phase of the viral infection, are associated with mortality in both solid organ transplant and immunocompetent COVID-19 patients.
While COVID-19 has a wide variety of clinical manifestations that range from mild respiratory symptoms to severe respiratory failure and death attributed to severe inflammation causing tissue damage and acute respiratory distress syndrome,
the mechanisms for disease progression during COVID-19 are yet not fully understood.
In the last decade, the potential of epitranscriptomics, or RNA epigenetics, has attracted great interest as a potential prognostic tool and novel therapeutic target for multiple diseases, such as cancer or cardiovascular disease. RNA modifications provide another layer of post-transcriptional modulation dynamically regulated by methyltransferases (writers), demethylases (erasers) and binding proteins (readers). Some recent data show that the well-studied epitranscriptomic modification N6-methyladenosine (m6A) impacts RNAs important for viral replication and host immune response to infection of many viruses, including SARS-CoV-2.
In fact, a study using sequencing data has shown that m6A in SARS-CoV-2 regulates the expression of genes associated with antiviral and inflammatory responses in lymphocytes, while in another study COVID-19 patients could be potentially stratified based on m6A profiling.
Though it is known that m6A in RNA is regulated by various proteins, data on the level of these proteins are missing in COVID-19 studies, which mostly report RNA transcript levels.
In this study, we examined protein and RNA levels of m6A regulatory proteins in relation to disease severity in a cohort of adults (mean age ± SD: 55 ± 15 years old; 11 males, 7 females) hospitalized with COVID-19 recruited during the first wave of the pandemic as previously described in the Norwegian SARS-CoV-2 study (NCT04381819).
COVID-19 severity was classified into non-respiratory failure (Non-RF) and respiratory failure (RF) according to the ratio PaO2/FiO2 (P/F) ≥ 37 kPa or< 37 kPa, respectively. For comparison, we also included data from gender-, age- and ethnicity-matched healthy controls (HC). Targeted-mass spectrometry and RNA-sequencing analyses were performed on peripheral blood mononuclear cells (PBMCs) from blood samples collected in BD CPT™ Cell Preparation Tubes containing sodium heparin (for more details, see Supplementary materials).
We explored relevant regulatory proteins associated with post-transcriptional modifications that catalyze methylation (writers) and demethylation (erasers), and bind (readers) to different types of RNA molecules that therefore may influence stability, location and translation of transcripts. We observed that the protein levels of the m6A-writers METTL3 and METTL5 were significantly lower in COVID-19 patients with RF as compared to HC (Fig. 1A). A recent study has reported that METTL3 transcript level is reduced in severe COVID-19 patients, and that the host METTL3 modified SARS-CoV-2′s genome, thus affecting viral replication, inducing inflammatory genes and evading the innate immune response in vitro.
In addition to this, we show that METTL3 is also reduced at a protein level in PBMCs of severe COVID-19. While METTL5 has not been previously linked to COVID-19, its ability to modulate cell stress response in preclinical models may be relevant for COVID-19.
Moreover, protein levels of the eraser FTO, known to interact with innate immunity, were significantly lower in patients with RF when compared to both Non-RF patients and HC (Fig. 1B). We observed that the levels of the reader eIF3A, which is part of the EIF3 translation initiation complex which influences T cell activation, were significantly higher in severely ill patients as compared to Non-RF patients and HC (Fig. 1C). We also investigated whether the levels of the regulatory proteins were associated with the degree of respiratory failure in COVID-19 (measured as P/F ratio). FTO and eIF3A protein levels showed a positive and negative correlation with P/F ratio, respectively (Fig. 1D). Of note, in contrast to our findings in PBMCs, Malbec et al. showed reduced FTO transcript levels negatively correlated to SARS-CoV-2 expression in lung epithelial cells from severe COVID-19 patients.
Fig. 1Relative protein levels of the m6A- (A) writers WTAP, METTL3, METTL14, METTL5 and DIMT1, (B) erasers FTO and ALKBH5, and (C) readers YTHDF2, YTHDF3 and eIF3A. Data is relative to GAPDH and presented as box and whiskers showing the minimum and maximum values of PBMC samples from healthy controls (HC; n = 11), non-respiratory failure COVID-19 patients (Non-RF; n = 12), and respiratory failure COVID-19 patients (RF; n = 6). Unpaired t-test performed for statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Panel (D) shows Pearson’s correlation analyses with 95% confidence interval of statistically significant (p < 0.05) findings between protein levels and P/F mean in COVID-19 patient samples.
Next, we explored RNA levels of the m6A-regulatory proteins. We observed that the RNA levels of the m6A-writer METTL14 were significantly downregulated in RF patients compared to HC, whereas ALKBH5 levels were lower in RF compared to both Non-RF and HC (Fig. S1A-B). Notably, neither METTL14 nor ALKBH5 was differentially regulated at protein level. Moreover, eIF3A transcript level was significantly lower in RF than in HC (Fig. S1C), whereas eIF3A’s was upregulated at protein levels. In addition, METTL14 and eIF3A RNA levels were positively correlated with RF (Fig. S1D).
The opposite trend of eIF3A in protein and RNA levels could be explained by self-regulation of eIF3A expression or could be influenced by the virus. Particularly, some studies have shown that SARS-CoV-2′s pathogenicity factor NSP1 can bind the host’s EIF3 translation initiation complex and block the translation of host transcripts.
Thus, the present findings also underscore the need for both protein and RNA data when evaluating the role of proteins regulating the epitranscriptome.
For the regulatory proteins of other important RNA modifications such as the m5C-writers NOP2 and NSUN5, the 2′O-Me writer FBL, and the m1A eraser ALKBH3 (Fig. S2A), we found no significant differences between groups. Yet, the RNA levels of NOP2 were significantly decreased in RF as compared to Non-RF patients and HC, and FBL levels were lower in RF as compared to HC (Fig. S2B).
In conclusion, we have shown that the levels of proteins regulating m6A methylation are altered in COVID-19 patients and associated with disease severity, highlighting potential roles for epitranscriptomic modifications in COVID-19. While larger studies that include new SARS-CoV-2 variants are needed to confirm this hypothesis, this represents potential new targets for improvement of therapies in COVID-19.
Funding
This study has been funded by: The Research Council of Norway with grant no. 295910, funding PROMEC as a member of the National Network of Advanced Proteomics Infrastructure via the INFRASTRUKTUR-program, and grant no. 312780; the South-Eastern Norway Regional Health Authority (Helse Sør-Øst RHF) with grant no. 2021071; and a private donation from Vivaldi Invest A/S owned by Jon Stephenson von Tetzchner.
Declaration of Competing Interest
Nothing to declare.
Acknowledgments
Mass spectrometry-based analyses were supported by PROMEC, Norwegian University of Science and Technology (NTNU) and The Central Norway Regional Health Authority.
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