Advances in the Structural and Functional Understanding of m1A RNA Modification

Abstract

ConspectusRNA modification is a co- or post-transcriptional process by which specific nucleotides are chemically altered by enzymes after their initial incorporation into the RNA chain, expanding the chemical and functional diversity of RNAs. Our understanding of RNA modifications has changed dramatically in recent years. In the past decade, RNA methyltransferases (MTases) have been highlighted in numerous clinical studies and disease models, modifications have been found to be dynamically regulated by demodification enzymes, and significant technological advances have been made in the fields of RNA sequencing, mass spectrometry, and structural biology. Among RNAs, transfer RNAs (tRNAs) exhibit the greatest diversity and density of post-transcriptional modifications, which allow for potential cross-talks and regulation during their incorporation. N¹-methyladenosine (m¹A) modification is found in tRNAs at positions 9, 14, 16, 22, 57, and 58, depending on the tRNA and organism.Our laboratory has used and developed a large panel of tools to decipher the different mechanisms used by m¹A tRNA MTases to recognize and methylate tRNA. We have solved the structures of TrmI from Thermus thermophilus (m¹A58), TrmK from Bacillus subtilis (m¹A22), and human TRMT10C (m¹A9). These MTases do not share the same structure or organization to recognize tRNAs, but they all modify an adenosine, forming a non-Watson-Crick (WC) interaction. For TrmK, nuclear magnetic resonance (NMR) chemical shift mapping of the binding interface between TrmK and tRNA^Ser was invaluable to build a TrmK/tRNA model, where both domains of TrmK participate in the binding of a full-length L-shaped tRNA and where the non-WC purine 13-A22 base pair positions the A22 N1-atom close to the methyl of the S-adenosyl-l-methionine (SAM) TrmK cofactor. For TRMT10C, cryoEM structures showed the MTase poised to N¹-methylate A9 or G9 in tRNA and revealed different steps of tRNA maturation, where TRMT10C acts as a tRNA binding platform for sequential docking of each maturation enzyme. This work confers a role for TRMT10C in tRNA quality control and provides a framework to understand the link between mitochondrial tRNA maturation dysfunction and diseases.Methods to directly detect the incorporation of modifications during tRNA biosynthesis are rare and do not provide easy access to the temporality of their introduction. To this end, we have introduced time-resolved NMR to monitor tRNA maturation in the cellular environment. Combined with genetic and biochemical approaches involving the synthesis of specifically modified tRNAs, our methodology revealed that some modifications are incorporated in a defined sequential order, controlled by cross-talks between modification events. In particular, a strong modification circuit, namely Ψ55 → m⁵U54 → m¹A58, controls the modification process in the T-arm of yeast elongator tRNAs. Conversely, we showed that m¹A58 is efficiently introduced on unmodified initiator tRNA_i^Met without the need of any prior modification. Two distinct pathways are therefore followed for m¹A58 incorporation in elongator and initiator tRNAs.We are undoubtedly entering an exciting period for the elucidation of the functions of RNA modifications and the intricate mechanisms by which modification enzymes identify and alter their RNA substrates. These are promising directions for the field of epitranscriptomics.

Figure 1

m¹A modifications in tRNAs and summary of available structural data. Schematic of a tRNA, showing the sites where m¹A modification can occur in red. The domains in which the modification has been identified are indicated with A (Archaea), B (Bacteria), and E (Eukarya). Some sites are specific to mitochondrial tRNAs, which are indicated with M. Structural data of m¹A9/m¹G9, m¹A22, and m¹A58 tRNA MTases are presented in the boxes with the methyltransferase domain (MTD) of the MTase in marine blue and their additional domain in cyan; places where a partner required for MTase activity are in gray. The endonuclease PRORP is in green, and the dashed blue circle indicates the interaction between the pentatricopeptide repeat (PPR) domain of PRORP and TRMT10C, which is required for RNase P activity of PRORP.

Figure 2

Temporality and circuit of modifications in pathways for m¹A58 introduction in yeast elongator and initiator tRNAs. (A) Overview of the methodological approaches used in combination for characterizing the temporality of modification incorporation and the potential modification circuits controlling their introduction. (B) Pathway for late m¹A58 incorporation in elongator tRNAs. The introduction of m¹A58 highly depends on the prior presence of Ψ55 and m⁵U54. Circles in red, blue, and purple represent modifications Ψ55, m⁵U54, and m¹A58, respectively. (C) Pathway for early m¹A58 incorporation in initiator tRNA_i^Met. NMR imino (¹H,¹⁵N) correlation spectra are shown next to unmodified- and m¹A58-tRNA_i^Met schematic drawings. In m¹A58-tRNA_i^Met, the U55 and G18 imino signals indicate that the elbow structure is properly folded.