Messenger RNA modifications: Form, distribution, and function

Abstract

RNA contains more than 100 distinct modifications that promote the functions of stable noncoding RNAs in translation and splicing. Recent technical advances have revealed widespread and sparse modification of messenger RNAs with N(6)-methyladenosine (m(6)A), 5-methylcytosine (m(5)C), and pseudouridine (Ψ). Here we discuss the rapidly evolving understanding of the location, regulation, and function of these dynamic mRNA marks, collectively termed the epitranscriptome. We highlight differences among modifications and between species that could instruct ongoing efforts to understand how specific mRNA target sites are selected and how their modification is regulated. Diverse molecular consequences of individual m(6)A modifications are beginning to be revealed, but the effects of m(5)C and Ψ remain largely unknown. Future work linking molecular effects to organismal phenotypes will broaden our understanding of mRNA modifications as cell and developmental regulators.

Fig. 1. Nucleotide modifications and detection strategies

For each modification, the chemical structure (left), detection strategy (right), and sample output of mapped sequencing reads (bottom) are shown. (A) For detection of m⁶A, antibodies are used to select methylated RNA fragments. A typical broad peak in read coverage overlaps an m⁶A site. nt, nucleotide. (B) Ψ are detected as CMC-dependent reverse transcriptase stops at the 3' end of the U site. (C) m⁵C is protected from conversion into U during bisulfite treatment, and putative sites are identified by their high rates of nonconversion. Each row represents one sequencing read, and filled squares indicate unconverted Cs. me, methylated site.

Fig. 2. Diverse molecular functions of m⁶A, Ψ, and m⁵C in coding RNAs

Nascent RNA transcripts in eukaryotic cells are chemically modified (red dot) by m⁶A, Ψ, and m⁵C “writer” enzymes. In the nucleus, m⁶A, and potentially other modifications, alters processing of pre-mRNA and pri-miRNA, through both direct recognition and induced changes in RNA secondary structure (,,). After export to the cytoplasm, which is enhanced by m⁶A (), mRNA modifications alter the efficiency and fidelity of translation (,,,,) and turnover of transcripts in the actively translating mRNA pool (,,).