The RNA Architecture of the SARS-CoV-2 3′-Untranslated Region

Figure A1. The workflow of DMS-MaPseq in this study. (a) BSR T5/7 cells were transfected with SARS-CoV-2 minigene for in vivo DMS-MaPseq. Infectious virions were collected for in-virion DMS-MaPseq. (b) Adenines and cytosines in the unpaired region of the transcriptome have a higher probability to be methylated by DMS. (c) The DMS-modified RNA was extracted. (d) The A- or C-methylated RNA can induce single-point mutations at the methylated nucleobase during the reverse transcription. Amplicons of 895 and 1291 base pairs were amplified by PCR for in-virion and in vivo minigene DMS-MaPseqs, respectively. (e) The band was purified by agarose gel electrophoresis. (f) The amplicons were ligated with Illumina sequencing adapters and sequenced in at a paired-end mode (150 bp × 2). (a,e,f) are generated by BioRender.com.

Figure 1. (a) The SARS-CoV-2 minigene construct containing the 5′ and 3′ untranslated regions (UTRs) and a short poly(A) tail. (b) The amplicon location (highlighted in yellow) in the SARS-CoV-2 genome for the in-virion DMS-MaPseq. The arrows indicate the primer-binding sites for the amplicons.

Figure 2. (a) The in vivo minigene, in-virion, and in vitro normalized dimethyl sulfate (DMS) activity (ΔnDMS) profiles of the SARS-CoV-2 3′ UTR. Compared to the in vivo minigene, the significantly higher or lower ΔnDMS activities are labeled in red and green, respectively (>95% confidence in Z-factor test and >1.5-fold ΔnDMS activity change, see Table S1 for details). The activity data are not available for the primer-binding regions, i.e., nts 319–327, 302–327, and 311–327 in the in vivo minigene, in-virion, and in vitro DMS-MaPseqs, respectively. (b) The correlations between the in vivo minigene and in-virion or in vitro ΔnDMS activities. The out-of-range data points are omitted in the figure. The in vitro DMS-MaPseq was re-analyzed using the data from Ref. [12] with the ShapeMapper2 pipeline (see Methods).

Figure 3. Arc plots of the most possible base-pairing pattern for SARS-CoV-2 3′ UTR. A colored arc represents a base pair with the following probability: 100% > green ≥ 80% > blue ≥ 30% > yellow ≥ 10%. By definition, ΔnDMS is high when ΔnDMS > 0.85, medium 0.40 < ΔnDMS ≤ 0.85, or low ΔnDMS ≤ 0.40, and the corresponding nucleotide symbol is colored red, orange, or black, respectively. The nucleotides of G and U, or within PCR primer-binding regions, where the DMS data is not available, are colored grey. The arc plots were generated by the SuperFold software package [26] from the experiments (a) in vivo minigene DMS-MaPseq, and (b) in-virion DMS-MaPseq. See Figure S2 for full arc plots with all possible base-pairing possibilities.

Figure 4. A consensus structure derived from the in-virion and in vivo minigene DMS-MaPseqs. The ΔnDMS reactivity for each nucleotide from the in vivo minigene DMS-MaPseq is colored according to the legend on the RNA backbone, with the in-virion DMS activity drawn aside if it belongs to a different reactivity group. The occurrences of the base pairing for BS-B–L1 and S3-A–S3-B (dotted lines) are not supported in this study.

Figure 5. (a) Two predominant conformations of bulged stem-loop (BSL) and stem-loop 1 (SL1) uncovered by the DREEM analysis on the DMS-MaPseq reads that cover >95% of the region. The illustrated structures were derived from replicate 2 data set. (b) Correlation in ΔnDMS activity profiles for the same structural cluster between the replicates and between two clusters.

Figure 6. Comparison of the 3′ UTR sequences among SARS-CoV-2 (RefSeq NC_045512.2), SARS-CoV (RefSeq NC_004718), MERS-CoV (NC_019843), BCoV (U00735), and MHV (NC_048217). Major RNA elements, BSL, SL1, and HVR, are boxed and annotated in black. Shorter RNA segments are annotated in blue above a bar aligned with the sequences. Conserved nucleotides are shaded in cyan. 717 nucleotides at the 5′ of MERS 3′ UTR are omitted for clarity.