To knot or not to knot: Multiple conformations of the SARS-CoV-2 frameshifting RNA element

  1. Tamar Schlick
  2. Qiyao Zhu
  3. Abhishek Dey
  4. Swati Jain
  5. Shuting Yan
  6. Alain Laederach

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Abstract

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 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 ribo-some 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.

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    SciScore for 10.1101/2021.03.31.437955: (What is this?)

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    Table 2: Resources

    Software and Algorithms
    SentencesResources
    MD simulations were performed using Gromacs 2020.4, 58 with the Amber OL3 forcefield.
    Gromacs
    suggested: (GROMACS, RRID:SCR_014565)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    While our predictions using 2D folding programs and chemical reactivity data do not identify other conformations besides the two pseudoknots and the 3-way junction, other possibilities cannot be excluded due to limitations in both the folding programs and reactivity experiments. Results for other works suggest other possible conformations, including two 3_8 pseudoknots, a different 3_3 pseudoknot (with Stem 2 formed by Stem 3 loop and 3′ end), a two-stem 2_1, and a two-stem-with-internal-loop 2_2 (Table S1 and Fig. S13). The length-dependent and context-specific conformations for the FSE emphasized in our work could be exploited biologically by the insidious virus in mechanisms of interactions with the ribosome. The bulky pseudo-knot may block and encourage ribosome pausing as the ribosome rolls over the FSE region, and any structural transitions may similarly interfere with this template-directed process. In the 2.3–7 Å-resolution Cryo-EM study by Bhatt et al., 21 the researchers observe the 3_6 pseudoknot wedged between the head and body of the small ribosomal subunit. Specifically, the residues before Stem 1 are found to be located at the mRNA entry tunnel so that, as translation proceeds, the GC-rich Stem 1 residues are unwound upon entering the mRNA tunnel. Stem 2 of the pseudoknot is found to be shorter by two base pairs than that predicted by computational 2D structure methods. To confirm these inferred structural interactions, the researchers mutated single residues i...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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  2. Version published to 10.1101/2021.03.31.437955 on bioRxiv