REVIEW ARTICLE The epitranscriptome in regulation: mRNA and tRNA modifications as the two sides of the same coin? Namit Ranjan1 and Sebastian A. Leidel2,3

1 Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany 2 Max Planck Research Group for RNA Biology, Max Planck Institute for Molecular Biomedicine, Munster,€ Germany 3 Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland

Correspondence Translation of mRNA is a highly regulated process that is tightly coordinated N. Ranjan, Department of Physical with cotranslational protein maturation. Recently, mRNA modifications and Biochemistry, Max Planck Institute for tRNA modifications – the so called epitranscriptome – have added a new Biophysical Chemistry, Goettingen, layer of regulation that is still poorly understood. Both types of modifications Germany – Tel: +49 5512012958 can affect codon anticodon interactions, thereby affecting mRNA translation E-mail: [email protected] and protein synthesis in similar ways. Here, we describe an updated view on S. A. Leidel, Department of Chemistry and how the different types of modifications can be mapped, how they affect Biochemistry, University of Bern, translation, how they trigger phenotypes and discuss how the combined action Bern, Switzerland of mRNA and tRNA modifications coordinate translation in health and Tel: +41 316314296 disease. E-mail: [email protected]

(Received 2 April 2019, revised 7 June Keywords: disease; epitranscriptome; post-transcriptional modifications; 2019, accepted 11 June 2019, available ; RNA modification; RNA sequencing; translation online 27 June 2019) doi:10.1002/1873-3468.13491

Edited by Maria Papatriantafyllou

Protein synthesis is essential to life. Thus, it is regulated ribosome during protein synthesis, proteins begin to by a variety of sophisticated mechanisms and comprises fold into their final three-dimensional structure and four phases: (a) initiation; (b) elongation; (c) termina- acquire protein modifications [2]. tion; and (d) ribosome recycling. Initiation is the key Some of the key players in translation like mRNA, regulatory step of translation, and different mechanisms tRNA or are RNA molecules or contain have been discovered that lead either to a global or tran- RNA molecules, essential to their function. These can be script-specific control of translation initiation [1]. How- post-transcriptionally modified by a plethora of chemical ever, translation initiation is not the only option for the modifications, which currently amount to more than a to regulate protein synthesis. Translation elongation hundred seventy [3–5]. Some of these RNA modifications comprises mRNA decoding, peptide-bond formation are evolutionary conserved and were linked to a human and tRNA–mRNA translocation, resulting in nascent- disease [6–8]. Since RNA modifications change the struc- peptide elongation, and each of these steps bears regula- tural and chemical properties of RNA molecules, some tory potential for translation. Furthermore, mRNA modifications of either mRNA, tRNA or rRNA are translation occurs in concert with protein maturation. thought to optimize translation dynamics in the cell, As soon as the nascent-peptide chain emerges from the whereas others may be neutral to translation [5].In

Abbreviations ac4C, N4-acetylcytidine; eIF, eukaryotic initiation factor; hm5C, 5-hydroxymethylcytosine; IP, immunoprecipitation; m5C, 5-methylcytosine; 6 6 6 6 0 5 m A,N-methyladenosine; m Am,N,2 -O-dimethyladenosine; RT, reverse transcription; SAM, S-adenosyl-L-methionine; sm U, 5-taurinomethyluridine; U34, wobble .

FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd 1483 on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. The epitranscriptome in translation regulation N. Ranjan and S. A. Leidel particular internal mRNA modifications and tRNA anti- us from understanding the in vivo roles of mRNA modi- codon modifications likely lead to very similar effects, fications and in particular from understanding their since the codon and the anticodon interact directly dur- effects during translation. ing decoding. Importantly, the reversibility of both types The most abundant internal mRNA modification is of modifications suggests a new, essentially unexplored m6A, which is present on average at 3 positions per layer of the control of that has been ter- mRNA molecule (Box 1). In addition to m6A, pseu- med the epitranscriptome [9,10].However,howparticu- douridine (Ψ), N1-methyladenosine (m1A), N6,20-O- lar mRNA or tRNA modifications affect translation is dimethyladenosine (m6Am), as well as m5C (Box 2) and largely unknown because the analysis of eukaryotic its oxidation product 5-hydroxymethylcytosine (hm5C), translation in vitro or in vivo and the identification of ac4C and 20-O-methylated nucleotides were mapped to internal mRNA modifications is very challenging. the . Additional modifications were Here, we discuss our emerging understanding of the reported based on measurements by RNA mass spec- role of mRNA modifications [N6-methyladenosine trometry but their position has not been determined (m6A), 5-methylcytosine (m5C), N4-acetylcytidine throughout the transcriptome [9,25]. The complex modi- (ac4C), 20-O-] and tRNA wobble uridine fication pattern of the transcriptome likely differs (U34) modifications in translation and how our under- between species and even between different tissues in standing of the roles of mRNA modifications is influ- one organism [26]. However, a complete map of all enced by current mapping approaches. Finally, we modifications in all cell types, still needs to be achieved comment on general implications for disease. This arti- and will require concerted efforts from many labs com- cle aims to convey a broad picture of how mRNA and parable to consortia like the ENCODE project [27]. tRNA modifications act in concert to control transla- tion. For a more focused discussion of mRNA and Internal mRNA modifications in tRNA modifications we refer the readers to several translational regulation recent reviews [9,11–20]. The recent identification of proteins that install (writ- 6 Internal mRNA modifications and their ers), recognize (readers) and remove (erasers) m A and mapping other modifications has revealed mechanisms how mRNA modifications can affect nearly every aspect of Internal modifications of mRNA and long noncoding the mRNA life cycle, as well as various cellular, devel- RNA have already been discovered in the 1970s [21–23]. opmental and disease processes. However, for under- However, due to their low abundance they generally standing their effects in translation, we currently have evaded biochemical analyses and we lacked the ability to mostly rely on biochemical and biophysical analy- to map their positions in a transcriptome-wide manner. ses. Using an in vitro protein synthesis kit containing mRNA modification mapping has become possible only the components for in vitro transcription and transla- recently, with the advent of deep sequencing and the tion, named ‘PUR-Express translation system’ with implementation of specialized sequencing-based proto- m5C, m6A, Ψ or 20-O-methylated nucleotides at each cols. In general, successful mapping approaches are position within a codon revealed that protein synthesis based on three strategies: (a) the induction or detection is strongly affected by nucleotide modifications in a of specific mutations in RNAseq experiments; (b) termi- position-specific manner [28].20-O-methylated nucleo- nation of reverse-transcription (RT) reactions and (c) tides at the 1st codon position affect translation only antibody-based enrichment of modification sites (exten- marginally; however, when placed at the 2nd position sively reviewed in [18]). Direct RNA sequencing appears the same modification causes an almost complete stop as a promising future alternative and has been shown to of protein synthesis. Introduction of multiple modified detect modified bases in synthetic RNA strands or nucleotides within one codon increased the translation abundant cellular targets [24]. However, these protocols inhibition [28]. More specifically, the presence of 20-O- have not been shown to work reliably on a transcrip- methylation at the 2nd codon position of mRNA tome-wide level, yet. It is important to realize that the strongly delays tRNA accommodation to the modified low abundance of mRNA modifications in combination codon [29]. Furthermore, 20-O-methylation impairs the with false-positive rates in their detection has remained initial and proofreading selection of aminoacyl-tRNA a challenge. Due to the lack of quantitative high-quality and the interaction between the codon–anticodon helix mRNA modification maps for most organisms we can- and ribosomal-monitoring bases [29]. Such alterations not easily correlate translational phenotypes to the of codon–anticodon interaction can even change the occurrence of mRNA modifications. This has prevented identity of the incorporated amino acid as the presence

1484 FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. N. Ranjan and S. A. Leidel The epitranscriptome in translation regulation

Box 1: Mapping of m6A sites

The mapping of m6A sites is the classical example for detection based on modification-specific antibodies [80,81]. Early protocols randomly fragment the mRNA and enrich m6A-containing mRNA fragments by immunoprecipitation (IP) using m6A-specific antibodies. The purified mRNA pool and a negative control are subsequently converted to cDNA and sequenced. Putative m6A sites are identified as m6A peaks that are absent in the control. While this strategy is sufficient to identify the position of m6A sites at a global level, it does not reach nucleotide resolution. Nucleotide resolution was later achieved by UV cross-linking the antibodies to mRNA prior to the IP called individual-nucleotide- resolution cross-linking and IP (miCLIP) [19,82,83]. The cross-link between RNA and the antibody leads to characteristic nucleotide substitutions that can be detected in the sequencing reaction. Antibody-based purification strategies have also been used for m1A, m6Am, hm5C and ac4C [18,19]. While m1A interferes with Watson–Crick basepairing, m6A is not distinguished from by standard RT . However, different strategies can be used to increase the mutation rates in such experiments like varying the concentration of dNTPs, using modified polymerases that are sensitive towards specific modifications or using selenium-modified deoxythymidine triphosphate analogues [84– 86]. An orthogonal strategy is to use analogues of S-adenosyl-L-methionine (SAM) [87]. Since SAM analogues are unstable under physiological conditions, cells are fed with propargyl-L-selenohomocysteine, which is converted to the respective SAM analogue by the cellular methionine adenosyltransferase [87]. Subsequently, cellular , which are often promiscuous towards their substrate use the SAM analogue and incorporate the methyl derivate into mRNA. The artificial label can then be used to enrich the labelled mRNA by chemical click reactions and to induce mutations and/or strand termination in the RT reaction [87].

strongly inhibits translation elongation dynamics [30]. 5 Box 2: Mapping of m C sites m6A acts as a barrier to tRNA accommodation when present at the 2nd codon position. However, when pre- 5 m C was the first modified nucleotide, which was mapped sent at the 3rd codon position of the near-cognate to the entire transcriptome after adapting bisulfite- codon m6A essentially does not affect tRNA selection conversion sequencing to RNA [17]. The method takes rate during translation elongation [30]. In addition, to advantage of the fact that methylation changes the the position of the modification, the sequence context chemical reactivity of . While acidic bisulfite also has a significant modulatory effect [28,30]. For 5 deamination converts cytidine to uridine, m C is resistant instance m6A in the glutamine (CAG) or proline to this conversion reaction. Therefore, the detection of (CCA) slowed translation elongation and Ψ at the 1st 5 m C relies on the identification of that do not position of the UAA increases transla- convert in response to bisulfite treatment. In identifying tional read-through [30,31]. However, translation elon- 5 thousands of m C sites throughout the transcriptome gation is not the only step that can be affected by RNA-bisulphite sequencing has become the first example nucleotide modifications. m6A like the cap, when pre- for identifying internal mRNA modifications in high sent in the 50 UTR of mRNAs can modulate transla- 5 throughput. However, the high number of m C sites has tion initiation. Even a single m6A nucleotide in the 50 been questioned, showing how much care needs to be UTR induces direct binding of mRNA to eukaryotic taken not to misinterpret modification marks that may be initiation factor 3 (eIF3), and is sufficient to recruit the result of single-nucleotide polymorphisms, inefficient the 43S complex to initiate translation in the absence chemical reactions or sequencing errors [20,88]. of the cap-binding initiation factor, eIF4E [32]. Fol- lowing heat shock, m6A was found more frequently in Hsp70 mRNA regulating its cap-independent transla- tion [32], an effect that appears to be mediated by ABCF1, a key factor in m6A-promoted translation of m5C at the 2nd codon position of CCC – a proline under both physiological and stress conditions [33]. 4 codon – leads to an amino acid-substitution by either Transcriptome-wide mapping of the ac C, catalysed 4 isoleucine or leucine [28]. by acetyltransferase NAT 10, revealed ac C enrich- A FRET-based assay using a bacterial translation ment within coding regions of mRNA [34]. Further- system revealed that m6A at the 1st codon position more, mRNA stability was decreased in NAT10-

FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd 1485 on behalf of Federation of European Biochemical Societies. The epitranscriptome in translation regulation N. Ranjan and S. A. Leidel deleted HeLa cells indicating that ac4C actively pro- ensured by the coordinated processes of transcription, motes mRNA stability and enhances translation. ac4C translation and maintenance of protein homeostasis. is strongly enriched in the 50 end of mRNA, however, During this process, tRNA modifications appear criti- no effect of ac4C on the formation of 48S preinitiation cal for maintaining this coordination [40–42]. In all complex was observed by in vitro analysis, demonstrat- known organisms wobble U34 carries a complex modi- ing that ac4C does not affect translation initiation [34]. fication at its 50 position and a 2-thio-group (s2U) in Finally, ribosome-profiling data show increased ribo- tRNALys(UUU), tRNAGln(UUG) and tRNAGlu(UUC). some occupancy for acetylated mRNA mediated by These tRNA decode A-ending codons of split codon NAT10, suggesting that ac4C intrinsically promotes boxes where U- and C-ending codons code for a dif- translation, a finding further supported in vivo by the ferent amino acid. Furthermore, U34 modifications quantification of translation products from parental were shown to be a determinant of efficient aminoacy- and NAT10-depleted cells [34]. lation of tRNAGlu and tRNAGln by increasing the Taken together, the emerging data suggest a direct binding affinity of the synthetase in Escherichia coli impact of internal mRNA modifications on gene but not in yeast [43–45]. On the ribosome, tRNA wob- expression, primarily through translation. ble modifications are implicated in maintaining accu- rate decoding and translational processivity. In 5 Ala(UGC) tRNA modifications and their mapping bacteria, cmo U34 in tRNA facilitates the decoding of codons ending with A, G and U, accord- tRNAs contain the largest diversity of modified ing to the wobble rules, but also the C-ending codon nucleotides [12,35]. Eukaryotic tRNAs contain on with reduced efficiency [46]. In tRNAGln(UUG) s2U34 average 13 modifications, including various methyla- affects the hydrolysis of GTP by EF-Tu and subse- tions, , dihydrouridine, thiolation and quently dipeptide formation [47]. Since the ratio others. Modifications are found throughout the tRNA between the rates of GTP hydrolysis and peptide bond molecule, with a hotspot in the anticodon loop. The formation is similar for s2U modified and unmodified modifications in the anticodon loop fine-tune decod- tRNAGln(UUG), it is likely that the modification prefer- ing, translational fidelity and translational efficiency, entially affects GTP hydrolysis and inorganic phosphate whereas tRNA modifications outside the anticodon (Pi) release with little effect on later steps in decoding loop mainly affect tRNA stability and modulate tRNA [47]. In , tRNALys(UUU), tRNAGln(UUG) and folding [12]. Furthermore, both classes of modifica- tRNAGlu(UUC) are decorated by 5-methoxycarbonyl- tions act as determinants for aminoacyl-tRNA syn- methyl-2-thiouridine (mcm5s2U34). The absence of thetase binding to tRNA. In recent years, numerous either of the two modifications leads to a codon-specific independent studies established an unexpected role of slowdown of translation of the AAA and CAA codons, tRNA modifications and the enzymes catalysing such which are decoded by tRNALys(UUU), tRNAGln(UUG) modifications for the aetiology of complex human [40,48]. GAA is not decoded more slowly in the yeast pathologies including , neurological and respira- mutant, consistent with the observation that reading- tory disorders and mitochondrial diseases [6–8,36]. frame maintenance of these codons depends on the Compared to mRNA, tRNA are very abundant. mcm5s2U modifications only for tRNALys(UUU) and Hence, their modifications have been characterized tRNAGln(UUG) [49] and that the overexpression of using RNA mass spectrometry and biochemical meth- tRNAGlu(UUC) is not able to rescue growth phenotypes ods, whereas similar methods were challenging for [50,51]. Hence, the absence of the modifications affects mRNA [37]. Nevertheless, the simultaneous quantifica- the global translation of a subset of mRNAs enriched tion of tRNA and their modifications in high through- for codons that are read by these tRNAs [52]. A com- put remains challenging, since sequence-specific RNA parison of the decoding properties of native modified mass spectrometry is not commonly used and some and unmodified tRNAs in an in vitro translation system RNA modifications perturb sequencing-based detec- showed that U34 modifications increase the affinity of tion. However, recent tRNA-sequencing protocols like the tRNA to its cognate codon in the A site of the ribo- ARM-Seq have improved the situation [38,39]. some [46,52]. Also the rate of peptide-bond formation at saturating concentrations of the ternary complex is 2 5 Wobble uridine in translational slower in the absence of s U34 or mcm U34 [52].A processivity real-time kinetic analysis shows that hypomodified tRNALys(UUU) that only carries mcm5U34 but lacks The ability of cells to respond and to adapt to dynami- s2U34 binds to its cognate codon with a lower affinity cally changing external conditions and stimuli are and is more frequently rejected than the fully modified

1486 FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. N. Ranjan and S. A. Leidel The epitranscriptome in translation regulation tRNALys(UUU). Nevertheless, the rate of peptide-bond Hypomodification and protein formation remains unaffected [53]. Altogether, these homeostasis data demonstrate a role of tRNA wobble uridine modi- fications at the initial steps of decoding and also during Modifications of tRNA optimize translation dynamics, the proofreading step [53]. which is crucial to maintain cellular homeostasis [40,60]. But how are the observed translational defects linked Several studies have characterized mutants that are defi- to phenotypes and to the regulation of gene expres- cient in wobble uridine modification using RNAseq, sion? Several studies have shown that 2-thiolation of ribosome profiling and quantitative proteomics tRNALys(UUU), tRNAGln(UUG) and tRNAGlu(UUC) is [40,48,52,54]. While no major translation defect was decreased at elevated temperature in yeast [54–57] and apparent based on S35 incorporation and polysome pro- that this effect is reversible when normal growth condi- filing [40,52], gene ontology analysis of downregulated tions are restored [56,57]. A study utilizing quantitative genes and proteins linked the loss of U34 modifications mass spectrometry and northern-blot analyses showed to processes like rRNA synthesis and processing, ribo- a decrease in s2U34 under permissive growth condi- some biogenesis, tRNA synthesis and modification, elec- tions upon nutrient starvation. However, thiolation tron transport chain and oxidative phosphorylation, pattern remained unaffected when sufficient nutrients and translation regulation, which are typically downreg- were supplied at elevated growth temperature, thereby ulated during the response to numerous stresses establishing that the reduction in 2-thiolation under [40,52,54,61,62]. Analyses of codon translation by ribo- heat stress is independent of nutrient availability [54]. some profiling in yeast cells lacking wobble uridine Under most circumstances, the lack of tRNA modifica- modifications revealed a seemingly mild enrichment of ~ tions induces cellular stress and negatively affects cell 20% of AAA and CAA codons in the A site of growth and survival [58]. Nevertheless, the absence of mutant ribosomes relative to wild-type [40,48,63]. This tRNA modifications can also be beneficial in specific is in agreement with a slight enrichment of AAA, CAA cases, for example by conferring resistance to endoplas- and GAA codons in mRNAs that appear reduced at the mic reticulum stress [55] or resistance to long-term proteomic level [52] and an in vitro study that showed that the absence of s2U34 affects the stability of nutrient starvation [59]. This indicates that the link Lys(UUU) between wobble uridine modifications and stress is tRNA binding to the ribosome during decod- more complex than it seems. ing and impedes rotation of ribosomal subunits upon

Fig. 1. Cellular outcomes of the presence or absence of tRNA anticodon modifications. Fully modified tRNAs ensure optimal translation through optimal decoding and translocation (left panel) resulting in a properly folded proteome. Slow decoding and translocation occurs due to loss of tRNA anticodon modifications (right panel), resulting in higher ribosome occupancy – indicative of a translation slowdown – at the codons requiring modified tRNAs and protein homeostasis defects.

FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd 1487 on behalf of Federation of European Biochemical Societies. The epitranscriptome in translation regulation N. Ranjan and S. A. Leidel

Fig. 2. tRNA modifications associated with human diseases. Schematic representation of a cytoplasmic tRNA with disease-linked modifications and the name of the associated diseases. tRNA–mRNA translocation [53]. The lack of s2U34 chaperones, responsible for cotranslational folding of modification increases the length of the decoding steps nascent polypeptides and preventing them from incor- and slows down translocation such that the residence rect folding [40,65]. Finally, the protein aggregates in time of the ribosomes on Lys-codon AAA increased wobble uridine modification mutants are not enriched by 20–40%, clearly accounting for the modest increase for AAA, CAA and GAA codons, but contain mainly in the ribosome occupancy revealed by ribosome pro- proteins that are known to be metastable [40,65]. filing [40,48,53]. Strikingly, however, the subtle trans- Taken together, the absence of tRNA modifications lation defects are accompanied by a perturbation of in induces seemingly mild effects on translation dynam- cellular protein homeostasis leading to the upregula- ics. However, those perturbations can significantly tion of cytoplasmic chaperones and the proteasome impact on cellular viability through its profound nega- and lead to the formation of protein aggregates in the tive effect on protein homeostasis. mutants [40] (Fig. 1). Similar phenotypes were observed in different modification mutants and may The epitranscriptome: implication in point to a common mechanism how defects in tRNA disease modifications induce phenotypes [41,64]. Interestingly, protein aggregates isolated from such cells are similar Mutations in nearly half of the RNA modification to those induced by the loss of ribosome-associated enzymes have been linked to human diseases, including

1488 FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. N. Ranjan and S. A. Leidel The epitranscriptome in translation regulation cancer, neurological disorders, genetic birth defects, under different conditions. However, most importantly cardiovascular diseases, mitochondrial-linked defects we will discover how those marks in trigger pheno- and metabolic disorders [8] (Fig. 2). Interestingly, types and facilitate physiological effects in different amongst the diseases that have been associated with species. An important step will be the further improve- mutations in RNA modifying enzymes, neurological ment of modification mapping. Deep-sequencing-based disorders are the most prevalent. This is in agreement methods have already made invaluable contributions with the observed enrichment of various RNA modifi- to our understanding of modified and the cations in neuronal tissues [66,67]. The m5C methyl- integration of additional enzymes and further improve- transferase NSUN2 has been associated with defects in ments of antibody-based strategies, will provide us memory and learning in Drosophila [68]. Furthermore, with novel insights. tRNA fragments were shown in NSUN2-deficient One of the major recent advances was the discovery mice, which may activate apoptosis in the brain [69]. how internal mRNA modifications affect gene expres- RNA demethylation has also been linked to neurologi- sion and translation. While most of the studies were cal defects. The deletion of the RNA demethylase performed using single mRNA modifications, it will be FTO in mice results in an impairment of dopamine very interesting to study the cross-talk between various receptor control of neuronal activity and behaviorial of this modification marks. Furthermore, it will be responses [70]. ALKBH5, a second m6A demethylase important to understand whether tRNA modifications has been associated to major depressive disorders [71]. play a role in decoding modified mRNA nucleotides In addition, mutations that affect 5-taurinomethy- and in particular how modified mRNA nucleotides luridine (sm5U) biosynthesis in mitochondrial tRNAs interact with tRNA anticodon modifications during are associated with mitochondrial diseases and affect translation. In the light of data highlighting the impact the translation of lysine and leucine codons. MTO1 of tRNA wobble modifications for translation quality and GTPBP3 catalyse the formation sm5U and through optimizing decoding, translocation and ribo- GTPBP3-knockout exhibit respiratory defects and some density, it appears worthwhile to explore the reduced mitochondrial translation, however, little is roles of other tRNA modifications. It is now well known about the mechanism [72–74]. established that gene expression regulation and human Defects in tRNA modifications and mRNA modifi- diseases are affected by tRNA modifications. It will be cations have also been directly tied to cell proliferation similarly important to understand which human and malignancy in a number of lymphomas, leukae- pathologies are linked to mRNA modifications. The mias and carcinomas, including breast, bladder and importance of RNA modifications for translation colorectal [6,75–77]. Elp3 and Ctu1/2, enzymes dynamics as well as their potential to perturb transla- responsible for mcm5s2 modification are upregulated in tion elongation will eventually tell us whether the two and were shown to be required to sustain types of modifications are indeed equal sides of the metastasis [78]. NSUN2 is a direct target gene of c- coin or whether translation is a coin with a flipside. Myc, a well-known proto- and has been found to be upregulated in primary tumours and Acknowledgements metastases of breast carcinomas [79]. The reduction in m6A levels through knockdown of Mettl3 results in Decades of work by many laboratories has uncovered tumour progression [77]. Taken together, these point to the roles of RNA modifications. In particular, wobble a crucial role of RNA modifications in human diseases. uridine modifications are amongst the most studied modifications. We are therefore, grateful to the scien- Concluding remarks tists in the field who have provided many important insights even though we have not been able to ade- This review aims to summarize the emerging view of quately reference their work due limited space. How- internal mRNA and tRNA modifications – the so ever, our review is also the result of discussions with called epitranscriptome – as regulators of translation numerous colleagues who have shared their insights and protein homeostasis. In the last decade we have and opinions. NR and SAL thank for the support by learned how RNA modifications – in particular in the the Deutsche Forschungsgemeinschaft (DFG) Priority tRNA anticodon and the newly discovered internal Programme SPP1784 Chemical Biology of native mRNA modifications – affect RNA metabolism in Nucleic Acid Modifications to NR (RA 3194/1-1) and unexpected ways, thus adding an unchartered layer to SAL (LE3260/2) and the Max Planck Society. SAL translation regulation. It will be exciting to follow how further acknowledges the support by DFG grant (LE new modifications will be mapped to the transcriptome 3260/3-1).

FEBS Letters 593 (2019) 1483–1493 ª 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd 1489 on behalf of Federation of European Biochemical Societies. The epitranscriptome in translation regulation N. Ranjan and S. A. Leidel

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