Fig 1.
Previously reported three-stemmed pseudoknots.
The three-stemmed pseudoknots were identified in different RNA molecules, including the frameshift stimulating pseudoknot at the ORF1a and ORF1b junction of the coronaviruses of SARS-CoV-1 and SARS-CoV-2 viruses (the two viral sequences have only one nucleotide difference C/A in the loop), as well as Infectious bronchitis virus (IBV), the frameshift stimulating pseudoknot at the gag-pol junction of HIV-1 subgroup-O viruses, the stop-codon readthrough stimulating pseudoknot in MoMuLV, the fluoride riboswitch, and the preQ1–II riboswitch. The numbering of the SARS-CoV-1/2, IBV, HIV-1, and MoMuLV pseudoknots is arbitrary, beginning with the first nucleotide of the displayed sequences. Every 10th nucleotide is indicated to assist in easily tracing the folding of the molecules.
Fig 2.
Computationally identified potential three-stemmed pseudoknots in human mRNAs, in close proximity to the AUG start codon.
The AUG start codon is highlighted in red. There is no intervening sequence between the stems, therefore the three stems (S1, S2 and S3) can potentially stack to form a quasi-continuous helix.
Fig 3.
Computationally identified potential multiple pseudoknots, including at least one three-stemmed pseudoknots, in the start codon region within human mRNAs.
The AUG start codon is highlighted in red. In the PK1 pseudoknots within the BPHL, SMIM10L2B and SMAP2 mRNAs, as well as the PK2 pseudoknot within the TMEM181 mRNA, there is no intervening sequence between the stems, therefore the three stems (S1, S2 and S3) can potentially stack to form a quasi-continuous helix.
Fig 4.
A) The three-stemmed pseudoknot in the CHD5 mRNA. The secondary structure and corresponding modeled three-dimensional structure are shown. The AUG start codon is heighted in red in the secondary structure. B) Structures of the −1 PRF stimulating three-stemmed pseudoknot in SARS-CoV-2 determined by X-ray crystallography (PDB code 7lyj and 7mlx) and cryoEM (PDB code 7o7z and 6xrz). The secondary structure revealed by 7lyj is also shown. Note that the apical loops in the two crystal structures are artificial. The structures show different base-paring schemes at the junction regions, which are also different from the originally proposed base-paring scheme as shown in Fig 1. The structures are color-ramped from 5’ in blue to 3’ in red.
Fig 5.
The three-stemmed pseudoknot in the CTDSPL mRNA.
The secondary structure and corresponding modeled 3-dimensional structure are shown. The AUG start codon is heighted in red in the secondary structure. The modeled structure is rendered in cartoon mode with color-ramping from blue at the 5′-end to red at the 3′-end.
Fig 6.
The tandem three-stemmed pseudoknots in the SMIM10L2B mRNA.
The secondary structure and corresponding modeled three-dimensional structure are shown. The AUG start codon is heighted in red in the secondary structure. The modeled structure is rendered in cartoon mode with color-ramping from blue at the 5′-end to red at the 3′-end.
Fig 7.
A proposed mechanism for the formation of the three-stemmed pseudoknot in the MAB21L4 mRNA.
Panel A outlines the sequential formation of this pseudoknot in three distinct steps, with further details provided in the main text. In Panel B, an alternative secondary structure for the same RNA sequence is presented, as predicted by RNAfold.