Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

Abstract

RlmN, a bacterial radical SAM methylating enzyme, has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet. Intriguingly, only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, suggesting that the enzyme carefully probes the highly conserved tRNA fold and sequence features to identify its targets. Over the past several years, multiple studies have addressed rRNA modification by RlmN, while relatively few investigations have focused on the ability of this enzyme to modify tRNAs. In this study, we utilized in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Using chimeras and point mutations, we probed how the structure and sequence of RNA influences methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition. We further demonstrate that, analogous to previous mechanistic studies with fragments of 23S rRNA, tRNA methylation requirements are consistent with radical SAM reactivity. Together, our findings provide detailed insight into tRNA recognition by a radical SAM methylating enzyme.

Fig 1. RNA methylation catalyzed by RlmN.

(A) RlmN catalyzes the conversion of adenosine (A) to 2-methyladenosine (m²A). RlmN modifies position A2503 in 23S rRNA (B) and position A37 in select tRNAs (C).

Fig 2. RlmN mediated methylation of in vitro transcribed tRNAs.

(A) End-point methylation of in vitro transcribed RNAs. Bars represent the mean of at least two replicates ± s.d. (B) Radio HPLC analysis of nucleosides obtained by methylation and total RNA digestion of select tRNA substrates. Radioactive signal co-elutes with an authentic m²A standard. (C) Evaluation of reaction requirements for methylation of in vitro transcribed tRNA^Gln_UUG. Bars represent the mean of two replicates ± s.d.

Fig 3. Interrogation of chimeric tRNA substrates.

(A) A representative 2D structure of tRNA. (B) In silico predicted representation of chimeric tRNA used in this study. Elements derived from substrate tRNA^Gln_UUG are in blue, while those deriving from non-substrate tRNA^Gly_CCC are in red. (C) End-point methylation of chimeric tRNA and tRNA^Gln_UUG ACSL RNA. Bars represent the mean ± s.d. of at least two replicates.

Fig 4. RlmN methylation of ACSL mutants.

(A) Sequence of tRNA^Gln_UUG ACSL. Numbers indicate the beginning and end of ACSL, as well as the target adenosine. (B) Alignment of ACSL regions from substrate (blue) and non-substrate (red) E. coli tRNAs. Darker colors represent a high degree of conservation. Lighter colors indicate that the nucleotide is less conserved. (C) End-point methylation of in vitro transcribed tRNA^Gln_UUG point mutants. Bars represent the mean of at least two replicates ± s.d.

Fig 5. Analysis of ACSL mutations in the context of tRNAGluUUC−RlmN C118A crystal structure [40].

Crystal structure of RlmN C118A in complex with tRNA^Glu_UUC showing the ACSL nucleotides U33 in yellow (A), U35 in green (B), C36 in magenta (C), and C38 in blue (D). Protein contacts between C38 (blue) and RlmN (cyan) are depicted in (E). In all panels, the target adenosine (A37) is depicted in red, the tRNA backbone in orange, the [4Fe-4S] cluster as red and yellow spheres, magnesium as a violet sphere, and RlmN in mint.