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To Knot or Not to Knot: Multiple Conformations of the SARS-CoV-2 Frameshifting RNA Element

The SARS-CoV-2 frameshifting RNA element (FSE) is an excellent target for therapeutic intervention against Covid-19. This small gene element employs a shifting mechanism to pause and backtrack the ribosome during translation between Open Reading Frames 1a and 1b, which code for viral polyproteins. Any interference with this process has a profound effect on viral replication and propagation. Pinpointing the structures adapted by the FSE and associated structural transformations involved in frameshifting has been a challenge. Using our graph-theory-based modeling tools for representing RNA secondary structures, “RAG” (RNA-As-Graphs), and chemical structure probing experiments, we show that the 3-stem H-type pseudoknot (3_6 dual graph), long assumed to be the dominant structure, has a viable alternative, an HL-type 3-stem pseudoknot (3_3) for longer constructs. In addition, an unknotted 3-way junction RNA (3_5) emerges as a minor conformation. These three conformations share Stems 1 and 3, while the different Stem 2 may be involved in a conformational switch and possibly associations with the ribosome during translation. For full-length genomes, a stem-loop motif (2_2) may compete with these forms. These structural and mechanistic insights advance our understanding of the SARS-CoV-2 frameshifting process and concomitant virus life cycle, and point to three avenues of therapeutic intervention.


FSE Mutation Counts
We downloaded the available British, South Africa, and Brazil variant sequences from GISAID on February 8, 2021, and 1000 randomly selected India variant sequences on July 8, 2021. We then aligned them with the reference following similar steps as GISAID's MSA: 1. Align each sequence to the reference by the following command line:

mafft --thread -1 [input] > [output]
2. Separate the sequences into two groups: Group 1 for sequences that bring insertions into the reference when performing Step 1 (G1 ), and Group 2 for the others (G2 ). 4. Add group 2 sequences into the alignment:
The recent highly transmissible British (B.1.1.7 or Alpha) variant is associated with 6 amino acid substitutions and 2 deletions in the spike protein. Among the 3575 British variant sequences, all of them have 5-11 mutations in the spike gene, while only 4 sequences have mutations (all single nucleotide) in the FSE segment. The mutation maps are shown in Fig. S2A. We find that 7 residues in the spike segment have mutation rates > 99.7%, and 6 of them correspond to the 6 amino acid substitutions. For the FSE segment, only 3 residues are mutated: C13506U twice, C13517U once, and A13533G once.
We conduct the same variant analysis for the recent concerning South Africa (B.1.351 or Beta) variant as well, and we see similar results. Among the 898 South Africa variant sequences, all of them have 4-12 mutations in the spike gene, while only 5 have mutations (all single nucleotide) in the FSE segment. In the mutation maps ( Fig.  S2B), 7 residues in the spike segment have mutation rates > 87.9%. In the FSE, 3 residues are mutated once, and the mutation C13517U seen above occurs twice.
Among the 94 Brazil (P.1 or Gamma) variant sequences, all of them have 9-13 mutations in the spike gene, while no mutation is seen in the FSE region (Fig. S2C). Moreover, 12 residues in the spike segment have mutation rates > 94.6%.
Finally, among the 1000 India (B.1.617 or Delta) variant sequences, all of them have 8-24 mutations in the spike gene, while only one has 2 mutations in the FSE segment. In the mutation maps (Fig. S2D), 18 residues in the spike segment have mutation rates > 99.5%. In the FSE, the two mutations are A13482G and C13517U, seen in South Africa variant as well.

Folding predictions for 4 pseudoknot-containing RNAs
To examine how sequence lengths affect 2D structure prediction programs, we selected four experimentally solved RNAs with pseudoknots from the Protein Data Bank (PDB) https://www.rcsb.org. The RNAs are: lysine riboswitch (PDB ID: 3DIO), lariat-capping ribozyme (6GYV), glmS ribozyme (2GCV), and T-Box riboregulator (6UFG). The 3D structures are extracted from respective PDB files, and corresponding 2D structures are determined using the RNApdbee webserver. 3 Pseudoknot substructures in these RNAs are identified and taken to define "short" systems for 2D structure prediction. An equal number of nucleotides are added to both ends of the lysine riboswitch and lariat-capping ribozyme pseudoknots to reach 120 nt. Only the 3 ends of the glmS ribozyme and T-Box riboregulator pseudoknot are expanded to 120 nt, because no upstream nucleotides are relevant. The five programs PKNOTS, NUPACK, IPknot, ProbKnot, and vsfold5 are used to predict 2D structures for three sizes ("short", 120 nt, and all). The predictions are compared to the 2D structures extracted from the experimental structures to assess the number of residues that are correctly base paired. The accuracies are calculated as the percentage of correctly base-paired residues in the total number of residues (Fig. S3).  Figure S3: Length effects on 2D structure predictions for four RNAs with pseudoknots. For each RNA, its experimentally determined 2D structure is shown, with the pseudoknot substructure boxed in red. Three length scales are taken for predictions using five programs: "short" for the pseudoknot substructure, 120 nt created by adding equal number of nucleotides on both sides of the pseudoknot, and "all" from the whole RNA structure. For the four RNAs as listed left to right, the pseudoknot lengths are 102, 106, 79, and 85 nt, respectively, and the total lengths are 174, 192, 151, and 166 nt, respectively. Prediction accuracies with respect to the experimental structures are determined by the relative number of correct base pairs for the three lengths, using PDB structures 3DIO for lysine riboswitch, 6GYV for lariat-capping ribozyme, 2GCV for glmS ribozyme, and 6UFG for T-box riboregulator. Although the accuracy varies with the method and RNA, the 120 nt window recommended in the literature appears reasonable in general.

Dual graph partition and substructure
We developed a partition algorithm that divides a dual graph into its component subgraphs while keeping pseudoknots and junction intact. 4 Here, we apply it to several important dual graphs mentioned in this paper (Fig. S4).
Dual graph 3 6 and 3 5 cannot be divided further, but graph 3 3 can be divided into 2 3, which represents the pseudoknot formed by intertwining Stems 1 and 2, and 2 1 representing two hairpins Stems 1 and 3. The two subgraphs are combined by overlapping one common vertex (Stem 1).
Dual graph 4 7 represents the two-pseudoknot structure predicted by NUPACK for the 84 nt FSE (Fig. 3), and it can be divided into 3 3 and 2 3. The 2 3 graph represents the extra pseudoknot formed by the loop region of Stem 3 and the 3 end.
Dual graph 4 12 represents the 3 6-containing structure predicted for the 87 nt FSE by ShapeKnots (Fig. S11), and here the 3 6 subgraph is detected by our partition algorithm. Meanwhile, this 4 12 is a subgraph of 6 132, which represents the 144 nt minor 3 6-containing structure (Fig. 4).