Abstract
N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.
Highlights
N6-methyladenosine (m6A) is an abundant reversible epitranscriptomic modification found in coding and non-coding RNAs [1,2,3,4]
Rev response element (RRE) is a ~350 nt cis-acting RNA element that is recognized by viral Rev protein to promote the export of unspliced or partially spliced viral RNA to express the structural proteins required for viral replication [22,23,24]
The two m6A sites were found in stem IIB (RREIIB), which is the primary binding site for Rev [23, 25,26,27]
Summary
N6-methyladenosine (m6A) is an abundant reversible epitranscriptomic modification found in coding and non-coding RNAs [1,2,3,4]. Of the two highly conserved adenines (A68) strongly suppressed viral replication (> 90%) [20] It was proposed [20] that the methyl group of m6A68 may interact with Rev protein to stabilize Rev-RRE binding, and/or that m6A68 may alter the conformational properties of stem IIB to facilitate Rev recognition. Another different study employing distinct cell lines and mapping methods did not observe these m6A sites on RREIIB suggesting that m6A can enhance HIV-1 replication and mRNA expression through recruitment of the m6A reader proteins YTHdomain containing family (YTHDF) [21]. While m6A has been shown to destabilize canonical duplexes [31, 32], it can stabilize junctional A-U base pairs in a Mg2+ and secondary structure dependent manner [37] as well as in contexts in which the m6A is in a dangling end [32]
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