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.

The y-ome defines the 35% ofEscherichia coligenes that lack experimental evidence of function

“…Some radical SAM enzymes are known to be involved in protein translation, e.g. rlmN ( 42 ). These observations provide some insight into the potential function of ygiQ .…”

miCLIP-MaPseq, a Substrate Identification Approach for Radical SAM RNA Methylating Enzymes

“…Our analysis shows that E. coli RlmN does not methylate tRNA Ala GGC , further confirming that this tRNA is not a substrate (Figure S3). Together, our results demonstrate that TGIRT-mediated mismatching is an efficient strategy to validate RlmN substrates and to identify sites on RNA modified by this enzyme with single-nucleotide precision. While TGIRT reverse transcribes protein scar with high efficiency (>80%) in most tRNA substrates, the efficiency of read-through at A37 decreases in the presence of adjacent bulky modifications (Table S3).…”

“…To further assess the specificity of the interaction between the mutant enzyme and its substrates we examined which tRNAs were enriched in the FLAG-tagged C118A RlmN sample. E. coli RlmN is known to modify only a subset of tRNAs that contain adenosine at the position A37. , Thus, for each tRNA we determined the fold change in its abundance between the FLAG-tagged C118A RlmN sample and the input control sample (Figure b). The control sample consisted of rRNA-depleted total RNA isolated from the E. coli BW25113 strain where endogenous RlmN was not FLAG-tagged; rRNA was depleted from the control total RNA to facilitate identification of low abundance RNAs.…”

The modification landscape ofP. aeruginosatRNAs

“…Reduced levels of m 1 G impair membrane structure and sensitize cells to antibiotics (Masuda et al 2019), suggesting a possible role for m 1 G in antibiotic resistance. m 2 A is deposited by RlmN (Benítez-Páez et al 2012; Fitzsimmons and Fujimori 2016). The majority of m 1 G and m 2 A sites had conserved signatures between P. aeruginosa and E. coli .…”