Chemical and structural effects of base modifications in messenger RNA

Abstract

A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification - the epitranscriptome - is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.

Figure 1. Structures of base-modified nucleosides known to be present at internal positions in mRNA

Six modified bases have been discovered so far: m⁶A, m¹A and inosine (top row) and m⁵C, hm⁵C and pseudouridine (bottom row). Chemical modifications are shown in red. Relevant purine and pyrimidine ring numbering is shown in blue.

Figure 2. Sequencing by chemical modification for the location of modified bases

a, Chemical treatments are applied to the modified bases pseudouracil and hypoxanthine, and the structures that result prevent the read through of RNA transcripts by reverse transcriptases. b, Sequencing by modification-specific termination of reverse transcription. The process for pseudouridine is shown as an example^–. Poly(A)⁺-enriched mRNA is fragmented and treated with CMC. A 3′-adaptor is ligated to each fragment and then reverse transcription is carried out. Next, truncated complementary DNA is selected by gel electrophoresis, amplified and sequenced. The location of the modification site is determined by comparing the frequency of read termination with and without the CMC treatment. c, The effect of sodium bisulfite (NaHSO₃) treatment on m⁵C and cytosine. The resulting structures are shown on the right. d, Bisulfite sequencing. Poly(A)⁺-enriched mRNA is fragmented and treated with sodium bisulfite. In the treated sample, all cytosines are converted to uracils; however, m⁵C is resistant to the treatment. The location of this base modification can be then identified as the sites that still code as cytosine after sodium bisulfite treatment. C, cytosine; U, uracil.