Fig. 3: Nucleotide substitutions and RBPs binding cause structural changes along SARS-CoV-2 genomes.

a Bar-chart summarizing nucleotide substitutions across all genome pairs of WT and VOC. b Box-plot comparing ΔSHAPE reactivity at conserved versus variable sites (normal n = 168,921, substitutions n = 76,128). The box indicates the first and third quartiles, and the median is indicated as the line. The whiskers show the minimum and maximum values, and the circles are outliers. Significance was calculated by two-sided t-test. c Top: Line plots showing structure change as a base transitions to either double- or single-stranded. Bottom: Line plot indicates the degree of structure similarity at each base centered on the substitution. Light blue shaded bands indicate ± variance in structure conservation around a genome position (n = 720). d, e RNA secondary structure models are constrained by SHAPE reactivity and visualized with VARNA for the indicated SARS-CoV-2 variant. Nucleotide substitutions are circled in green. f Bar graph illustrating the number of differentially expressed RBPs in all VOC pairs. g Upset plot integrating six studies to define the SARS-CoV-2 RNA-bound proteome (RBPome). h Venn diagram depicting the intersection of several RBP sets from multiple databases. Confirmed SARS-CoV-2 RBPs are unique RBPs aggregated from literature sources, while Prospective - (DGE RBPs) are differentially expressed genes cross-referenced with Confirmed and ATtRACT Database RBPs lists. i Scatterplot correlating RBP expression with local structure similarity around RBP binding motifs. Experimentally confirmed RBPs are  in light blue (Confirmed), while predicted RBPs from motif enrichments are coloured in black (Prospective). j Structure models of a region containing an HNRNPU motif (circled in purple) in WT, Beta, and Omicron show conserved stem-loops with reorganized long-range interactions. Structure changes are concordant with variant-specific HNRNPU expression. k Top, ΔSHAPE profile of HNRNPU binding region on SARS-CoV-2 genome comparing WT-infected with Beta-infected cells. Bottom, ΔSHAPE profile for an in vitro 500-nt fragment probed in the presence and absence of HNRNPU protein. Significantly less structured regions (positive change) are in light blue (two-tailed paired t-test, p < 0.01). HNRNPU motif is boxed in purple. Source data are provided as a Source Data file.