Dynamic RNA Modifications in Gene Expression Regulation

Abstract

Over 100 types of chemical modifications have been identified in cellular RNAs. While the 5' cap modification and the poly(A) tail of eukaryotic mRNA play key roles in regulation, internal modifications are gaining attention for their roles in mRNA metabolism. The most abundant internal mRNA modification is N⁶-methyladenosine (m⁶A), and identification of proteins that install, recognize, and remove this and other marks have revealed roles for mRNA modification in nearly every aspect of the mRNA life cycle, as well as in various cellular, developmental, and disease processes. Abundant noncoding RNAs such as tRNAs, rRNAs, and spliceosomal RNAs are also heavily modified and depend on the modifications for their biogenesis and function. Our understanding of the biological contributions of these different chemical modifications is beginning to take shape, but it's clear that in both coding and noncoding RNAs, dynamic modifications represent a new layer of control of genetic information.

Figure 1. Chemical modifications in eukaryotic messenger RNA

A schematic representation of common chemical modifications in eukaryotic mRNA transcripts. Several of these modifications map uniquely to the mRNA cap structure, 5’ or 3’ untranslated regions, or the coding region (bold) of the transcript.

Figure 2. Active m⁶A methylation, demethylation, and downstream consequences for protein-RNA interactions

(A) m⁶A is installed co-transcriptionally by a complex consisting of METTL3, METTL14, WTAP, and KIAA1429. Each of these components binds mRNA and is required for complete methylation, but only METTL3 contributes to the catalytic activity of the complex. (B) m⁶A methylation affects protein-RNA interactions through multiple mechanisms. Methylation can perturb the secondary structure of mRNA, exposing or masking potential RNA-binding motifs (top). Selective m⁶A-binding proteins exhibit increased affinity for methylated mRNAs, and in turn incorporate these transcripts into various steps of mRNA metabolism (middle). Methylation itself introduces hydrophobic moieties. In the case of m⁶A, association with hydrophobic amino acid side chains or low complexity regions of proteins may assist in solvation of the modified base (bottom).

Figure 3. RNA modification groups transcripts for cellular processes

Developmental programs require rapid switching of the cellular transcriptome to bring about phenotypic changes. Recruitment of transcription factors can alter the composition of the RNA pool by introducing required transcripts to the existent mRNA population. In order to accomplish more rapid transitions, cells utilize the existing pool of mRNA, tuning their expression accordingly. One way to differentiate distinct groups of mRNAs from a diverse cellular pool is by post-transcriptional modification by m⁶A, which marks transcripts for incorporation into pathways for translation and decay. Additional modifications may lend greater diversity to such mechanisms.

Figure 4. Landscape of tRNA and rRNA modifications

(A) All annotated mammalian tRNA modifications were pulled from Modomics (http://modomics.genesilico.pl/). Blue residues have no modification annotated while yellow residues are modified in at least one mammalian cytosolic tRNA (Bos taurus Homo sapiens, Mus musculus, Oryctolagus cuniculus, Ovis aries, Rattus norvegicus). The anticodon, a hot spot of modification, is highlighted in red. m¹A58, the first tRNA modification shown to be reversible, is circled. Modifications that occur in the variable loop were left out for simplicity. m²G -N²-methylguanosine; m²₂G - N²N²-dimethylguanosine; m¹G - 1-methylguanosine; ac⁴C - N⁴-acetylcytidine; m¹A - 1-methyladenosine; D - dihydrouridine; acp³U - 3-(3-amino-3-carboxypropyl)uridine; m³C - 3-methylcytidine; I - inosine; m¹I - 1-methylinosine; mcm⁵U - 5-methoxycarbonylmethyluridine; mcm5s2U - 5-methoxycarbonylmethyl-2-thiouridine; Q - queuosine; galQ - galactosyl-queuosine; manQ - mannosyl-queuosine; f⁵Cm - 5-formyl-2’-O-methylcytidine; t⁶A- N6-threonylcarbamoyladenosine; ms²t⁶A - 2-methylthio-N6-threonylcarbamoyladenosine; m⁶t⁶A - N⁶-methyl-N⁶-threonylcarbamoyladenosine; i⁶A - N⁶-isopentenyladenosine; o²yW - peroxywybutosine; yW - wybutosine; m¹Ψ - 1-methylpseudouridine; Ψ_m - 2’-O-methylpseudouridine; m⁷G - 7-methylguanosine; m⁵C - 5-methylcytidine; m²A - 2-methyladenosine; m⁵U - 5-methyluridine. B) All annotated Homo sapiens rRNA modifications recorded in Modomics (http://modomics.genesilico.pl/) were mapped onto the cryo-EM structure of the human ribosome (4ug0). An E- site tRNA (green) is shown for orientation. 2’-O-methylations are shown in blue, pseudouridylations are shown in yellow, and base modifications are shown in red. For simplicity, 2’-O-methyl-seudouridine is shown in blue. The rRNA ribbon diagram shows the prevalence of modification (~2% of total RNA residues). As the surface rendering incorporating ribosomal proteins shows, the large majority of the modifications are buried within the core of the ribosome.

Figure 5. Mechanisms of selectivity in m⁶A installation and downstream regulation

Mechanisms for selectivity in installation of m⁶A are largely unknown, and cannot be predicted by primary sequence alone. Potential mechanisms include recruitment of methyltransferase components to nascent RNA by chromatin features associated with RNA Polymerase II (green), or exclusion from the transcription complex (red). RNA-binding proteins that occupy consensus sequences for m⁶A may also prevent installation of the mark (orange). Once methylated, transcripts can be bound by RNA-binding that recognize modifications or secondary structure changes, or actively demethylated and no longer subject to regulation by m⁶A-dependent pathways. Transcripts that are heavily decorated with methylations may face large solvation penalties, and benefit from trafficking within RNA granules.