Colocalization of m⁶A and G-Quadruplex-Forming Sequences in Viral RNA (HIV, Zika, Hepatitis B, and SV40) Suggests Topological Control of Adenosine N⁶-Methylation

Abstract

This Outlook calls attention to two seemingly disparate and emerging fields regarding viral genomics that may be correlated in a way previously overlooked. First, we describe identification of conserved potential G-quadruplex-forming sequences (PQSs) in viral genomes relevant to human health. Studies have demonstrated that PQSs are highly conserved and can fold to G-quadruplexes (G4s) to regulate viral processes. Key examples include G4s as a countermeasure to the host’s immune system or G4-guided regulation of replication or transcription. Second, emerging data are discussed concerning the epitranscriptomic modification N 6 -methyladenosine (m 6 A) in viral RNA installed by host proteins in a consensus sequence favoring 5′-GG(m 6 A)C-3′. The proposed pathways by which m 6 A is written, read, and erased in viral RNA genomes and the impact this has on viral replication are described. The structural reason why certain sites are selected for modification while others are not is still mysterious. Finally, we discuss our new observations regarding these previous sequencing data that identify m 6 A installation within the loops of two-tetrad PQSs in the RNA genomes of the Zika, HIV, hepatitis B, and SV40 viruses. We hypothesize that conserved viral PQSs can provide a framework (sequence and/or structural) for m 6 A installation. We also discuss literature sources suggesting that PQSs as sites of RNA modification could be a general phenomenon. We anticipate our observations will provide ample opportunities for exciting discoveries regarding the interplay between G4 structures and epitranscriptomic modifications of RNA.

Potential G-quadruplex forming sequences andN6-methyladenosine colocalize at human pre-mRNA intron splice sites

“…Our prior interest in the colocalization of m 6 A and PQSs focused on viral RNA genomes that showed a preference for DRACH motif methylation. 10 The present work on human pre-mRNA is consistent with the viral RNA analysis; however, the sequence context found for the colocalization in intronic pre-mRNA occurs largely within SAG motifs. This difference observed may reflect the writing of m 6 A on pre-mRNA occurs in the nucleus, while m 6 A in viral RNA occurs in the cytosol.…”

“…[1][2][3][4][5] This has led to the suggestion that RNA secondary structure plays a key role in selection of m 6 A modified sites. [9][10][11] Additional epitranscriptomic modifications include pseudouridine (Ψ) and A-to-I editing yielding inosine (I; Figure 1A), in which similar lingering questions regarding the sites selected for modification have been asked. 9 Alternative splicing of nascent or pre-mRNA to yield mature mRNA is a highly regulated eukaryotic process resulting in a single gene being able to code for multiple proteins.…”

“…In RNA, stable rG4 folds have been found with only two G-tetrads, which is in contrast to DNA that generally requires at least three G-tetrads to adopt a stable fold. 10,15 The greater stability in rG4s results from the 2′-OH providing an additional hydrogen bond that is not possible in DNA. 15 Prior studies have shown that two-tetrad rG4s provide a quasi-stable fold that can be harnessed as a switch to impact the fate of RNA in cells.…”

“…23 The finding of m 6 A and PQS colocation in human mRNA, particularly introns of pre-mRNA, suggests that the rG4 secondary structure and epitranscriptomic m 6 A may be synergistic; this observation is consistent with our previous report on a similar finding in viral genomic RNA. 10 Whether the G4 fold is the signal for writing m 6 A in the mRNA or the presence of m 6 A impacts the G4 fold is not known. 10 The statistical significance for enrichment of PQSs in the regions of m 6 A enrichment in the human mRNA was compared to randomized, shuffled sequences ( Figure 2C).…”

“…10 Whether the G4 fold is the signal for writing m 6 A in the mRNA or the presence of m 6 A impacts the G4 fold is not known. 10 The statistical significance for enrichment of PQSs in the regions of m 6 A enrichment in the human mRNA was compared to randomized, shuffled sequences ( Figure 2C). Comparison of the 23,372 PQSs identified in the HEK293 pre-mRNA to the 5,461 PQSs expected by randomized shuffling found a 4.3-fold enrichment in PQSs; this finding is significant on the basis of Fisher's exact test (P < 2.2 e -16 ; Figure 2C and Table S2).…”