SARS-CoV-2 remodels the landscape of small non-coding RNAs with infection time and symptom severity

Abstract

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has significantly impacted global health, stressing the necessity of basic understanding of the host response to this viral infection. In this study, we investigated how SARS-CoV-2 remodels the landscape of small non-coding RNAs (sncRNA) from a large collection of nasopharyngeal swab samples taken at various time points from patients with distinct symptom severity. High-throughput RNA sequencing analysis revealed a global alteration of the sncRNA landscape, with abundance peaks related to species of 21-23 and 32-33 nucleotides. Host-derived sncRNAs, including microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), and small nucleolar RNA-derived small RNAs (sdRNAs) exhibited significant differential expression in infected patients compared to controls. Importantly, miRNA expression was predominantly down-regulated in response to SARS-CoV-2 infection, especially in patients with severe symptoms. Furthermore, we identified specific tsRNAs derived from Glu- and Gly-tRNAs as major altered elements upon infection, with 5' tRNA halves being the most abundant species and suggesting their potential as biomarkers for viral presence and disease severity prediction. Additionally, down-regulation of C/D-box sdRNAs and altered expression of tinyRNAs (tyRNAs) were observed in infected patients. These findings provide valuable insights into the host sncRNA response to SARS-CoV-2 infection and may contribute to the development of further diagnostic and therapeutic strategies in the clinic.

Fig. 1. Experimental design and bioinformatic analysis.

a Overview of the experimental design, in which sample acquisition is followed by extraction, preparation, and sequencing of sncRNAs. b Bioinformatics pipeline for analysis of sncRNA sequencing data. c Principal component analysis based on sRNA accumulation. d Histogram showing the relative accumulation (and distribution) of the total clean reads of sRNAs ranging from 12 to 34 nt from the libraries analyzed. Controls and different patient groups are represented with colors. Error bars correspond to standard errors. RPM reads per million. Error bars represent mean ± standard error of the mean.

Fig. 2. Global landscape of altered sRNAs upon SARS-CoV-2 infection.

a Graphic representation of the expression values (DESeq2) of sRNA sequences for the different conditions against control samples. Each dot corresponds to a given sRNA expression value. Colors indicate significant differential expression with |log2FC | > 0.585 and FDR < 0.05 for the different sRNA families (sRNAs with non-significant differential expression are in gray). The category Other is used to label those endogenous sRNAs that could not be annotated or were annotated as miRNAs with a length not compressed between 19 and 24 nt, lncRNAs, rRNAs, scaRNAs, protein-coding or miscellaneous RNAs. sRNAs annotated as derived from microorganisms are colored in light brown. b Detail of the number of sequences differentially expressed for each sRNA family in the four conditions analyzed. miRNA micro RNA, tsRNA tRNA-derived small RNA, snsRNA small nuclear-derived RNA, sdRNA small nucleolar-derived RNAs, tyRNA tiny RNA and piRNA: Piwi-interacting RNA.

Fig. 3. Expression levels of different miRNA families with consistent differential expression in the four groups analyzed.

Boxplot representation (on the top, patients with severe symptoms; on the bottom, patients with moderate symptoms). Each dot represents the expression value (log2FC) of a sequence in infected patients (T1 or T2) with respect to the non-infected control. In boxplots, the central lines depict the median, while the box boundaries represent the upper and lower quartiles. The whiskers extend to the first or last data point within 1.5x the interquartile range of the box boundaries in the lower and upper directions, respectively.

Fig. 4. Expression profiles of altered Glu-tRNA- and Gly-tRNA-derived sRNAs for each condition.

Four classes of tRNA-derived sRNAs are represented: 5’tRF, 3’tRF, 5’tR-half, and 3’tR-half. Dots indicate the absolute accumulation in reads per million (RPM) of differentially expressed sequences classified as Glu-tsRNA or Gly-tsRNA in control and infected samples for each condition (severe/moderate symptoms, T1/T2 time). In boxplots, the central lines depict the median, while the box boundaries represent the upper and lower quartiles. The whiskers extend to the first or last data point within 1.5x the interquartile range of the box boundaries in the lower and upper directions, respectively.