Biological Function and Sequencing Technologies of m1A RNA Modification

Introduction to N1-methyladenosine (m1A) Sequencing

In eukaryotic cells, N1-methyladenosine (m1A) is found in thousands of different gene transcripts (from yeast to mammals). The majority of m1A methylation modifications occur in the 5'UTR region of mRNA, and it is a common post-transcriptional modification in tRNA and rRNA in eukaryotes. m1A at the first and second nucleotides of the transcript promotes mRNA translation, whereas m1A in the coding region inhibits it. In mammalian cells, m1A also plays an active and dynamic role in the initiation of translation, according to recent research. m1A can influence the balance between alternative and canonical translation initiation sites by regulating the intensity of specific translation initiation sites. m1A sequencing (m1A-seq), a technique that relies on the immunoprecipitation of m1A-containing RNA fragments, provides a comprehensive view of m1A modifications. The idea is to co-incubate randomly interrupted RNA fragments with m1A RNA methylation-specific antibodies, capture methylated fragments, and then deep sequence them to get complete maps of m1A sites in the transcriptome. m1A-seq can detect m1A methylation in RNAs at a high rate, allowing for a better understanding of mRNA transcription and translation, as well as the structure and function of tRNA and rRNA. m1A-seq aids in the comprehension of the role and function of this novel epigenetic transcriptome marker.

Figure 1. Reversible RNA Modification N¹-methyladenosine (m¹A) in mRNA and tRNA. (Zhang, 2018)

m1A Modification in tRNA

The m1A modification occurs in cytosolic (cyt) tRNA at five different positions (9, 14, 22, 57, and 58), two of which are also found in mitochondrial (mt) tRNAs. The m1A modification in nucleotide position 14 (m1A14) is uncommon and has only been found in (cyt) tRNAPhe from mammals, whereas the m1A22 modification has only been found in bacterial tRNAs. The m1A57 modification was discovered in archaea and occurs only transiently as an intermediate in the hydrolytic deamination of 1-methylinosine (m1I). The m1A58 modification is found on all three domains of life's (cyt)tRNAs, as well as (mt)tRNAs. The m1A9 modification can be found in archaeal (cyt)tRNA or mammalian (mt)tRNA.

Biological Role of m1A Modifications in tRNA

Both the well-studied m1A9 and m1A58 modifications have been connected to tRNA structural stability andor proper folding. This link has been studied in depth for the m1A9 modification in human (mt)tRNALys, which subsists in vitro as an extended hairpin structure in equilibrium with the classical and functional tRNA 'L-shape' conformation. By interrupting a destabilizing Watson–Crick interaction that would otherwise form between A9 and U64, the m1A9 modification transitions this equilibrium towards the 'L-shape' structure. Because the A9–U64 interaction has been disrupted, U64 can now bind to A50, allowing the tRNA to maintain its functional L-shaped structure. Structural plasticity was also confirmed for (mt)tRNALeu(UUR) and (mt)tRNAAsp, implying that post-transcriptional modifications may influence the fold of these (mt)tRNAs, similar to what was seen for (mt)tRNALys.

The structural thermostability of tRNA has been linked to the m1A58 modification. When m1A58 was combined with two other post-transcriptional modifications (Gm18 and m5s2U54), the melting temperature of tRNAs from Thermus thermophilus increased by about 10 °C, compared to the unmodified transcript, and the absence of the enzyme that forms m1A58 resulted in thermosensitivity in bacterial tRNAs. The m1A58 gene in human tRNALys3 has been shown to be critical for the fidelity and efficiency of reverse transcription by retroviruses such as HIV-1. m1A58 has also been linked to the maturation of the yeast initiator tRNAMet.

References:

1. Ali AT, Idaghdour Y, Hodgkinson A. Analysis of mitochondrial m1A/G RNA modification reveals links to nuclear genetic variants and associated disease processes. Communications biology. 2020 Mar 27;3(1).
2. Zhang C, Jia G. Reversible RNA Modification N1-methyladenosine (m1A) in mRNA and tRNA. Genomics, proteomics & bioinformatics. 2018 Jun 1;16(3).
3. Oerum S, Dégut C, Barraud P, Tisné C. m1A post‐transcriptional modification in tRNAs. Biomolecules. 2017 Mar;7(1):20.