ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

Abstract

High-throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA methylation sequencing (ARM-seq), which uses pretreatment with Escherichia coli AlkB to demethylate N(1)-methyladenosine (m(1)A), N(3)-methylcytidine (m(3)C) and N(1)-methylguanosine (m(1)G), all commonly found in tRNAs. Comparative methylation analysis using ARM-seq provides the first detailed, transcriptome-scale map of these modifications and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-seq demonstrates that tRNA fragments accurately recapitulate the m(1)A modification state for well-characterized yeast tRNAs and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state and may reveal new roles for tRNA fragments as biomarkers or signaling molecules.

Figure 1. ARM-Seq facilitates sequencing of m¹A, m³C, or m¹G modified RNAs

AlkB-facilitated RNA methylated sequence (ARM-Seq) uses enzymatic demethylation of RNA samples prior to RNA-seq library preparation to reveal RNAs containing m¹A, m³C, or m¹G. Widely used protocols for small RNA sequencing, including NEBNext (New England Biolabs) and TruSeq (Illumina), require ligation of sequencing adapters to both the 5′ and 3’ ends of each RNA prior to reverse transcription for library preparation. Without any additional treatments, sequencing output from these protocols will therefore represent only RNAs with appropriate end chemistry for sequencing adapter ligations (5′-monophosphate and 3′-OH, the expected end chemistry of mature tRNAs, some classes of tRNA-derived fragments, microRNAs, and snoRNAs) that produce full-length cDNAs. “Hard-stop” modifications such as m¹A, m³C or m¹G, which commonly occur in tRNAs, cause premature termination of cDNA synthesis, preventing PCR amplification and subsequent sequencing. Typical positions for these modifications are indicated in the schematic showing tRNA secondary structure in canonical cloverleaf form. In ARM-Seq, removal of m¹A, m³C, or m¹G modifications by AlkB treatment facilitates the production of full-length cDNAs from previously modified templates, producing a ratio of reads in treated versus untreated samples that can be used to identify methylated RNAs.

Figure 2. ARM-Seq reveals m¹A-modified tRNA fragments in S. cerevisiae

(a) ARM-Seq more than doubled the fraction of yeast small RNA sequencing reads mapping to tRNAs, revealing a diversity of methylated small RNAs derived from tRNAs. The majority of these were 3′-fragments and half-molecules of tRNAs, where m¹A at position 58 (m¹A₅₈) is the most prevalent hard-stop modification. Full-length tRNAs comprised less than 1% of tRNA reads in both AlkB-treated and untreated samples, consistent with a known bias in sequencing library preparation where 5′ linker ligation is impeded by recessed 5′ ends of mature tRNAs. (b) ARM-Seq read profiles show increases in 3′-fragment reads relative to untreated samples that predict the presence of m¹A₅₈ in Thr-AGT, Leu-GAG and Gln-TTG (indicated by *). By contrast, ARM-Seq profiles for Arg-CCG, Gly-CCC and His-GTG show comparable or diminished 3’ reads for untreated samples, predicting un-modified A₅₈ in these tRNAs. (c) Primer extensions targeting the corresponding mature tRNAs demonstrate that these ARM-Seq results reflect the modification patterns of mature tRNAs, confirming the A₅₈ modification state documented in Modomics for Thr-AGT and His-GTG, providing new information on the m¹A₅₈ modification state of Arg-CCG, Gly-CCC and Leu-GAG tRNAs, and presenting new evidence that Gln-TTG tRNAs contain m¹A₅₈. (d) As a genome-scale screen, ARM-Seq correctly predicts m¹A₅₈ modification state for yeast tRNAs with accuracy of 94% as corroborated by documentation in Modomics, or verification by primer extension (for tRNAs indicated in red), based on increases of two-fold or more (dotted red line) and P < 0.01 (indicated by *).

Figure 3. ARM-Seq reveals methylated RNAs derived from human cytosolic tRNAs, tRNA precursors, and mitochondrial tRNAs

(a) Transcriptome-scale screening using ARM-Seq provides evidence for m¹A₅₈ modification in a majority of human tRNA isotypes, showing a consistent profile of modification in two B-cell derived human cell lines (with * indicating significant responders). (b) Profiles for many tRNA-derived small RNAs revealed by ARM-Seq show little, if any detection in untreated samples, indicating high levels of modification. (c) ARM-Seq also provides the first evidence that many human pre-tRNAs are m¹A₅₈ modified at an early stage prior to removal of 5’ leader and 3’ trailer sequences from primary transcripts (demarcated by dashed lines), demonstrating the ability to resolve sequential modification and processing steps involved in tRNA maturation. The 5′-leader sequences of these precursor-derived RNAs were typically short (4–5 nt) when present, which might reflect either nucleolytic processing or dephosphorylation of triphosphorylated primary transcripts to generate 5’-monophosphate ends (required for RNA-seq library inclusion). By contrast, the 3′-trailers were often 9–10 nt or longer, frequently ending with a poly-U sequence, suggesting that these represent the 3′-ends of primary RNA polymerase III transcripts. Reads for full-length and fragmentary pre-tRNAs revealed by ARM-Seq included the T-loop region, consistent with m¹A₅₈ modifications. (d) Fragments of human mitochondrial tRNAs revealed by ARM-Seq demonstrate a capacity to also demethylate m¹A₉ (in mito-Asp-GTC, mito-Lys-TTT), m¹G₉ (mito-Ile-GAT), and m¹G₃₇ (mito-Leu-TAG, mito-Pro-TGG), enabling investigation of mitochondrial diseases related to tRNA modification and processing. tRNAs for which ARM-Seq predictions were verified by primer extension are indicated in red.