FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA

Abstract

Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

TRMT2B is responsible for both tRNA and rRNA m5U-methylation in human mitochondria

“…On the basis of mitochondrial localization and the loss of m 5 U54 in mt-tRNAs in TRMT2B-ablated cells, this work concludes that TRMT2B is responsible for the m 5 U54-methyltransferase activity in human mitochondria. By extension, this work corroborates an earlier finding that TRMT2A is responsible for this activity in the cytosol [23]. In addition, the presence of m 5 U429 in 12S mt-rRNA, predicted from its detection in hamster mitochondria, is supported by this study, and also appears to be a product of TRMT2B.…”

“…This approach was repeated for a selection of other mt-tRNAs containing U54, out of which mt-tRNA Asn and mt-tRNA Gln also appeared to contain m 5 U54 ( Figure S3). In agreement with the previously identified role for TRMT2A 23 , the m 5 U54 status of cytosolic tRNAs were found to be unaffected following the loss of TRMT2B activity ( Figure S4). Collectively, these results show TRMT2B is a mitochondrial protein responsible for the modification of position 54 in mt-tRNAs.…”

“…The m 5 U54 modification has been identified in both cytoplasmic and mitochondrial tRNAs in humans [25], with the former recently identified to be catalysed by TRMT2A [23], one of the two human orthologs of yeast Trm2 along with TRMT2B. As a mitochondrial role for TRMT2A was not ruled out by Carter et al [23]., we set out to identify which of these paralogs may be operating within human mitochondria.An alignment of protein sequences from a range of species shows that the SAM binding site is highly conserved in the TRMT2A and TRMT2B paralogs. Whilst this is also the case for the remainder of the active site in TRMT2A, TRMT2B displays some dramatic divergences in some species (Fig.…”

“…Efforts to characterize the modification profile of human tRNAs have identified the presence of m 5 U54 in the majority of cytoplasmic tRNAs as well as a small number of mitochondrial tRNAs. A very recent study demonstrated that human TRMT2A is responsible for introduction of the majority of m 5 U in human RNA, and that it targets U54 of cytosolic tRNAs [23]. The enzyme responsible for m 5 U in human mt-tRNA however remains to be characterized, although TRMT2B has been previously suggested as a candidate [24].…”

Epitranscriptomics of Mammalian Mitochondrial Ribosomal RNA

“…While invertebrates possess a single representative of this family, Trm2, responsible for the cytosolic tRNA modification [ 131 ], vertebrate genomes encode its two paralogues, TRMT2A and TRMT2B. TRMT2A is capable to modify cytosolic tRNA [ 134 ], while TRMT2B became specialized not only in mitochondrial tRNA modification, but also in modification of the 12S rRNA. This evolutionary history is reminiscent to the emergence of the dual specificity methyltransferase TRMT61B.…”