# Advances in Human Pre-tRNA Maturation: TRMT10C and ELAC2 in Focus

Juhi Sikarwar, Vincent Meynier, Carine Tisné

► To cite this version:

Juhi Sikarwar, Vincent Meynier, Carine Tisné. Advances in Human Pre-tRNA Maturation: TRMT10C and ELAC2 in Focus. Journal of Molecular Biology, 2025, 437 (16), pp.168989. 10.1016/j.jmb.2025.168989. hal-05294114

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# Advances in Human Pre-tRNA Maturation: TRMT10C and ELAC2 in Focus ☆

Juhi Sikarwar, Vincent Meynier, and Carine Tisné*

Université Paris Cité, CNRS, Institut de Biologie Physico-Chimique (IBPC), Microbial Gene Expression Unit, 75005 Paris, France

Correspondence to Carine Tisné: carine.tisne@cnrs.fr (C. Tisné)
https://doi.org/10.1016/j.jmb.2025.168989

Edited by Jane E. Jackman

## Abstract

Mitochondrial pre-tRNA maturation is a multi-step process involving the removal of the 5'-leader by PRORP, 3'-trailer processing by ELAC2, 3'-CCA addition by TRNT1, and the incorporation of post-transcriptional modifications. In metazoans, the low structural stability of mitochondrial pre-tRNAs adds significant complexity to these steps, and defects in their maturation have been implicated in various human mitochondrial disorders. In this case, the tRNA methyltransferase complex TRMT10C/SDR5C1 compensates for the pre-tRNA structural alteration to present the pre-tRNA to maturation enzymes. Cryo-electron microscopy structures of human mitochondrial pre-tRNA maturation complexes have provided critical insights into these essential processes. Here we review the current understanding of tRNA maturation within human mitochondria and explore its implications for nuclear pre-tRNA maturation.

© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Transfer RNAs (tRNAs) are essential for protein synthesis, where they play the role of adaptor molecules between messenger RNAs (mRNAs) and elongating peptide chains on active ribosomes. They are thus central to the decoding process, by delivering amino acids corresponding to the mRNA sequence. To achieve this key task, they must adopt a three-dimensional structure as an L-shape, formed by a combination of stacking interactions and conserved tertiary interactions between the D-, T- and variable loops, to interact with most of the components of the translation apparatus. This structure is universally shared by all tRNAs, even mitochondrial (mt) tRNAs, which sometimes exhibit very “weird” secondary structures. Indeed, mt-tRNAs have low structural stability compared to cytosolic tRNAs because

they have a low GC content and their D-, T- and variable loops, which support the folding of cytosolic tRNAs, are either absent or differ in length.¹ For instance, in humans, mt-tRNA Ser (AGY) has no D-arm at all, while the D-loop of mt-tRNA Cys and the T-loop of tRNA Thr contain only three nucleotides compared to an average of 11 and 7 nucleotides, respectively, for cytoplasmic tRNAs.

The three-dimensional L-shaped structure of tRNAs results from a complex and multi-step maturation process. The biogenesis of a mature tRNA begins with the transcription of a tDNA gene to produce a precursor tRNA (pre-tRNA) that has to be matured to remove the 5'-leader and the 3'-trailer by RNase P and RNase Z, respectively, and to add the 3'-CCA amino-acid accepting sequence at its 3'-end.²⁻⁴ When present, introns are also spliced out during the maturation process. Each of these stages also involves chemical modifications of nucleotides.⁵ tRNAs are the RNA family that exhibits both the greatest diversity of modifications

<footnote>* This article is part of a special issue entitled: 'tRNA modifications (2025)' published in Journal of Molecular Biology.</footnote>
tions and the greatest number of modifications per molecule.⁶,⁷ For instance, about 6 modifications are found on average in human mitochondrial tRNAs, with each tRNA having its own specific combination of the 18 chemically distinct modifications that occur in 137 locations in the human mt tRNAs.⁸ The purine-9N'-methylation, which occurs in 14 tRNAs on an adenosine (m¹A9) and for 5 tRNAs on a guanosine (m¹G9), is the most prevalent modification in these tRNAs.⁸ TRMT10C, which belongs to the Trm10 family of tRNA methyltransferases (MTases) found in archaea and eukaryotes, catalyzes these m¹A9 and m¹G9 modifications in human mt-tRNA, using S-adenosyl-L-methionine (SAM) as the methyl donor cofactor.⁹⁻¹¹ Thus far, TRMT10C is the only member of the Trm10 family which requires a partner to be active: the protein SDR5C1 belonging to the short chain dehydrogenase/reductase family of proteins.¹²,¹³,⁹ Besides this tRNA MTase activity, TRMT10C/SDR5C1 subcomplex has been shown to be involved in mt-tRNA maturation steps essential for mt-translation, i.e. 5'-leader and 3'-trailer processing, and 3'-CCA addition of the structurally-degenerate mitochondrial pre-tRNAs in metazoans.⁴,¹¹,¹⁴

Like their bacterial ancestors, mitochondria have retained a circular double-stranded genome (mt-DNA). All mt-DNA genes have a singular purpose: the translation of proteins in the respiratory chain to generate OXPHOS activity. In humans, the mt-DNA encodes 37 genes: 13 essential proteins of the respiratory chain, 2 mt-ribosomal RNAs (mt-rRNAs), and 22 mt-tRNAs.¹⁵,¹⁶ All RNA components required for mt-translation are thus supplied by mitochondria, whereas all the protein components are encoded by the nuclear genome, translated in the cytoplasm and imported into the mitochondria. Each mt-DNA strand is transcribed as a large polycistronic transcript.¹⁷ In all metazoan species, the organization of mt-DNA follows the 'punctuation model', where mt-tRNA genes are located between rRNA and mRNA genes.¹⁶,¹⁸ The sequential 5'- followed by 3'-end processing of mt-tRNAs by mt-RNase P (PRORP – protein-only RNase P- or MRPP3)¹² and mt-RNase Z (ELAC2),¹⁹ respectively, releases the rRNAs and most of the intervening mRNAs from the polycistronic transcripts.¹⁶,¹⁸,²⁰,¹¹,²¹ Regions within the polycistronic transcripts that lack tRNAs are processed by FASTK-family members.²²,²³ Processed mt-tRNAs further require the addition of the 3'-CCA sequence by TRNT1²⁴ for amino-acid charging by aminoacyl-tRNA synthetases, and the incorporation of nucleotide chemical modifications to become fully mature and functional for mt translation. Impaired processing of mt-tRNAs has a detrimental effect on mt-gene expression and assembly of mt-ribosomes.²⁰,²¹ As a consequence, mutations that disturb mt-tRNA structures or any step of mt-tRNA maturation, including mutations either on

TRMT10C, PRORP or ELAC2, are linked to severe human diseases.¹²,²⁵⁻²⁹

The molecular mechanisms of tRNA-end processing and 3'-CCA addition have been recently visualized by cryo-electron microscopy (cryoEM) for the mt-RNase P in complex with pre-tRNA<sup>Tyr14</sup> or with the pair of pre-tRNA<sup>His</sup>-tRNA<sup>Ser</sup>(AGY),⁴ for the mt-RNase Z with various states of ELAC2,<sup>4,30-32</sup> and TRNT1 in complex with mt-tRNA<sup>Ile</sup>.⁴ Comprehensive reviews on mt-RNase P are already available.³³,³⁴ Here, we review our current understanding of polycistronic-transcript processing and tRNA maturation in human mitochondria with a specific focus on the Trm10 and RNase Z enzyme families.

## Human mt-tRNA maturation steps supported by TRMT0C/SDR5C1

CryoEM structures of human mitochondrial tRNA maturation steps at high resolution (2.7–3.2 Å) have shown how the methyltransferase complex, TRMT10C/SDR5C1, serves as a maturation platform that presents the mt-pre-tRNA to maturation enzymes (PRORP, ELAC2, and TRNT1, Figure 1a–d).⁴ The organization of the maturation complexes with mt-tRNA is globally the same in all structures. TRMT10C straddles a tetramer of SDR5C1 while wrapping around the pre-tRNA, which adopts an L-shaped structure (Figure 1). In all structures, a second copy of TRMT10C/pre-tRNA is bound on the opposite side of the SDR5C1 tetramer as also described for the mt-RNase P/pre-tRNA<sup>Tyr</sup> complex,¹⁴ confirming the 2:4 TRMT10C:SDR5C1 stoichiometry previously reported.¹⁰,¹²,¹⁴ The second copy displayed less clear densities, likely due to lower occupancy of TRMT10C, preventing its complete modeling. However, TRMT10C has been found in two different orientations as schematized in Figure 1e.¹⁴ This suggests that two pre-tRNAs could be matured simultaneously, independently of the maturation stage of either (Figure 1e).

TRMT10C interacts with the pre-tRNA stabilized in an L-shaped conformation, even though the T- and D-loops do not interact. A long helix in the N-terminal domain (NTD) of TRMT10C establishes interactions from the anticodon- to the T-loops, maintaining this L-shaped conformation like a stake. This arrangement exposes the acceptor and T-arms of the mt-pre-tRNA, particularly the T-loop, for interactions with the maturation enzymes (Figure 1). Additionally, all nucleotides of the anticodon loop interact with conserved residues of the TRMT10C NTD. This domain also interacts with the maturation enzymes. Indeed, our mt-RNase P structure (PDB 8CBK) reveals parts of TRMT10C invisible in the previously published complex with pre-tRNA<sup>Tyr</sup> (PDB 7ONU, Supplementary Figure 1), notably an interaction
A purine-9 N'-methylation

<figure><img src="image_3.png" /></figure>

B mt-RNase P

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C mt-RNase Z

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D 3'-CCA addition

<figure><img src="image_6.png" /></figure>

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E

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F

<figure><img src="image_12.png" /></figure>

**Figure 1.** Molecular machineries responsible for human mitochondrial tRNA processing and 3'-CCA addition. (A) TRMT10C/SDR5C1 in complex with mt-tRNA<sup>Ile</sup> (PDB 8CBO), only the catalytic domain of TRMT10C is visible. (B) TRMT10C/SDR5C1 in complex with mt-tRNA<sup>His</sup>-tRNA<sup>Ser(AGY)</sup> and PRORP for 5'-leader processing (PDB 8CBK). (C) TRMT10C/SDR5C1 in complex with mt-tRNA<sup>His</sup>-tRNA<sup>Ser(AGY)</sup> and ELAC2 for 3'-trailer processing (PDB 8CBL). (D) TRMT10C/SDR5C1 in complex with mt-tRNA<sup>Ile</sup> and TRNT1 for 3'-CCA addition (PDB 8CBM). (E) Schematic showing the architecture of the maturation complexes taking into account that two TRMT10C molecules can bind to the SDR5C1 tetramer (in grey), one on each side in a parallel or anti-parallel orientation of TRMT10C molecules (in green). (F) Superimposition of maturation complexes with PRORP (in cyan) and with ELAC2 (in marine) showing that PRORP and ELAC2 cannot simultaneously bind the pre-tRNA.

with the PPR domain of PRORP and TRMT10C NTD (Figure 1B, dashed circle). This interaction is required for PRORP to perform 5'-leader cleavage.⁴

The processing must be sequential, because PRORP and ELAC2 cannot bind to the pre-tRNA simultaneously (Figure 1f). Moreover, ELAC2 cannot bind a tRNA with unprocessed 5'-leader, whereas PRORP binding involves interactions with the 5'-leader and 3'-trailer,⁴ highlighting the

molecular mechanisms of the sequential tRNA-end processing presented in Figure 2. First, TRMT10C and SDR5C1 form a subcomplex, which can bind and methylate the pre-tRNA. PRORP is either already bound to the pre-tRNA or recruited by the TRMT10C/SDR5C1/pre-tRNA subcomplex to catalyze the 5'-cleavage. The 3'-cleavage by ELAC2 and 3'-CCA addition can then occur through a factor exchange, first, between PRORP and
<figure><img src="image_13.png" /><figcaption>**Figure 2.** Molecular basis of the human mitochondrial pre-tRNA maturation process. The TRMT10C/SDR5C1 complex binds mt-pre-tRNAs and presents them as substrates for nucleolytic cleavages at defined 5' and 3' sites by PRORP and ELAC2, respectively, and finally for 3'-CCA addition by TRNT1.</figcaption></figure>

ELAC2, and then between ELAC2 and TRNT1 (Figure 2). The mitochondrial tRNA methyltransferase subcomplex can thus be viewed as a factor that compensates for the structural variability in the mitochondrial tRNA substrates and provides an additional attachment point for the maturation enzymes to guarantee proper maturation of structurally degenerate pre-tRNAs.⁴ Metazoan mitochondrial tRNAs exhibit low structural stability and variability in conserved features, such as the elbow formed by interactions between the D- and T-loops. Unlike other RNase P, RNase Z, and 3'-nucleotidyl transferase enzymes that rely on the conserved tRNA elbow for interactions,³⁵⁻³⁸ mitochondrial enzymes primarily engage with the T-loop of pre-tRNAs and the TRMT10C NTD (Figure 2). This interaction is required for PRORP activity,⁴ but is not necessary for ELAC2 or TRNT1, as both enzymes remain active on pre-tRNAs, whether or not they are bound to the TRMT10C/SDR5C1 complex.⁴,¹¹ However, TRMT10C/SDR5C1 facilitates cleavage by ELAC2 for some pre-tRNAs.¹¹,³² Indeed, the differential dependence of pre-tRNAs

on 3'-processing by ELAC2 is related to the tRNA elbow region, whether it is formed or not. TRMT10C compensates for the absence of canonical tRNA elbow structure in a subset of human mitochondrial pre-tRNAs to provide fully mature mt-tRNAs required for mt-translation.

A key unresolved question is the status of pre-tRNAs following PRORP processing, specifically, whether they remain bound to the TRMT10C/SDR5C1 complex or are released. N'-methylation, catalyzed by TRMT10C, does not trigger tRNA release.¹¹,³⁹ *In vitro* studies have shown that pre-tRNAs remain bound to the TRMT10C/SDR5C1 complex after PRORP and ELAC2 processing and even after TRNT1 3'-CCA addition, with evidence of no multiple enzymatic turnovers.¹¹,³⁹ Structural analyses show that the TRMT10C/SDR5C1 complex serves as a scaffold, presenting mitochondrial pre-tRNAs as substrates for chemical modification, end processing, and 3'-CCA addition.⁴ However, the mechanism that governs dissociation of tRNAs from the methyltransferase complex remains unknown.
In the following paragraphs, we explore the insights gained into the Trm10 family of methyltransferases and RNase Z enzymes.

## Trm10-enzyme family across evolution

tRNA MTases of the Trm10 family are found in archaea and eukaryotes. In archaea, the crenarchaeal *Sulfolobus acidocaldarius* Trm10 forms m¹A9⁴⁰ and the euryarchaeal *Thermococcus kodakaraensis* Trm10 displays a dual specificity, forming m¹A9 and m¹G9 in tRNAs.⁴⁰,⁴¹ Metazoa appear to have duplicated the trm10 gene, with up to three different Trm10 paralogs encoded in vertebrates (Figure 3). Besides TRMT10C, two additional enzymes from the Trm10 family, TRMT10A and TRMT10B, are expressed in humans. TRMT10A (the direct homolog of yeast Trm10) and TRMT10B modify cytoplasmic (ct) tRNAs (Figure 3). Each human Trm10 enzyme N1-methylates a unique subset of tRNAs, modifying only G9 of a subset of ct-tRNAs for TRMT10A,⁴²⁻⁴⁴ only A9 of ct-tRNA⁵⁵⁸p for TRMT10B,⁴²⁻⁴⁴ or exhibiting dual specificity to modify either G9 or A9 of mt-tRNAs for TMRT10C.⁴⁵ TRMT10C also catalyzes a single m¹A modification in the ND5 mt-mRNA.⁴³,⁴⁶

The situation is further complicated by the fact that, the enzymes Trm10 in yeast and TRMT10A in humans, introduce the m¹G9 modification in approximately half of the cytosolic tRNAs having a G at position 9 *in vivo*, in contrast to what is observed *in vitro*.⁴⁴,⁴⁷⁻⁴⁹ The *in vivo* tRNA substrates of yeast Trm10 lack a common sequence or post-transcriptional modifications that could explain why only some G9-containing tRNAs are selected for modification. A notable feature of non-substrate tRNAs for SPOUT-fold MTases is the long variable loop of type-II tRNAs.⁵⁰ The molecular basis for *in vivo* substrate selection by Trm10 and TRMT10A thus remains poorly understood.

Most structural studies of Trm10-family enzymes have been limited to complexes with SAM or SAH.⁴¹,⁵¹,⁵² An exception is the TRMT10C structure shown in Figure 1,⁴ which reveals TRMT10C in complex with both SAH and tRNA. These structures capture the pre-catalytic states for m¹A9 and m¹G9 reactions, where A9 and G9 are flipped and stabilized into the TRMT10C active site by the same set of conserved residues for methylation (N222, Q226, D314, and N350). The active site adopts the characteristic SPOUT-fold of Trm10-family enzymes, defined by a trefoil knot that forms a pocket for SAM binding, positioning the methyl group for efficient transfer. The aspartate residue D314 is positioned to form a hydrogen bond with the N6 amino group of A9, which triggers an SN2 mechanism.⁴ For G9, no residue is ideally positioned to deprotonate the N1 atom; however, D314 remains the best candidate, and its mutation abolishes TRMT10C activity.¹⁰ For S. cerevisiae Trm10, *H. sapiens* TRMT10A, and *T. kodakarensis*

Trm10, the lack of a conserved residue that could act as a required general base to deprotonate G9 was explained by a rate-determining ionization of G9 using a collaborative active site that facilitates the transfer of the N1 proton to water. This ionization is not necessary to methylate A9 nucleotides, resulting in a distinct pH dependence for this reaction.⁴⁰,⁵²,⁴¹,⁵³,⁴⁸ Finally, the asparagine N348 side chain could participate in the dual specificity of TRMT10C through a conformational switch that allows to accommodate a guanosine or an adenosine in the TRMT10C active site.⁴

The methyl transfer reaction in Trm10-family enzymes involves base flipping of the target nucleotide into the enzyme's active site.⁴ This suggests that tRNA flexibility, or the enzyme's ability to induce specific conformational changes in the tRNA upon binding, could account for differences in substrate specificity observed in vivo versus in vitro. Recent SHAPE structure probing analysis revealed distinct conformational changes in S. cerevisae Trm10-substrate tRNAs required for methylation, changes not observed in non-substrate tRNAs bound to Trm10 in vitro.⁴⁹ These include increased reactivity in the D-loop and decreased reactivity in the anticodon loop of substrate tRNAs.⁴⁹ CryoEM structures and binding assays show that the TRMT10C NTD functions as an RNA-binding domain.⁴ It includes a long α-helix that interacts with the anticodon, variable, and T-loops, presenting the T-loop to maturation enzymes. Comparisons with predicted structures of Trm10-family enzymes in complex with tRNAs confirm that their NTDs adopt a similar fold. The conserved long α-helix interacts with regions from the anticodon loop to the T-arm (Figure 3). This interaction likely explains the reduced anticodon loop reactivity in substrate tRNAs,⁴⁹ while the α-helix likely changes D- and T-loop interactions, consistent with increased D-loop reactivity. In archaeal Trm10 enzymes, this α-helix is absent. Instead, additional domains with unrelated folds bind tRNAs (Figure 3). While TRMT10A and TRMT10B have an NTD comprising a single long α-helix, TRMT10C's NTD is more complex, comprising additional α-helices involved in binding maturation enzymes, at least in humans.⁴ The extended NTD of TRMT10C and its requirement for a partner protein are likely specific to Trm10 enzymes modifying mitochondrial tRNAs in bilaterians. This adaptation is necessary because mitochondrial tRNAs have lost key secondary structure elements that are conserved in cytoplasmic tRNAs.

## Structural basis of RNase Z activity

RNase Z enzymes, found across all domains of life, belong to the zinc-dependent metallo-β-lactamase family⁵⁵ and exist in two forms: short (RNase ZS) and long (RNase ZL). RNase ZS, present in prokaryotes and eukaryotes, functions as a
<figure><img src="image_14.png" /><figcaption>**Figure 3.** Structural prediction of tRNA recognition across Trm10 family. The models were generated using either Alphafold<sup>54</sup> or by aligning yeast tRNA<sup>Phe</sup> structure (PDB 6LVR) and Alphafold3 models<sup>54</sup> of the Trm10-family enzymes with the structure of TRMT10C bound to tRNA<sup>His</sup>-tRNA<sup>Ser</sup>(AGY) (PDB 8CBK). The Trm10 homologues are represented in green and bound to tRNA in red. For Archaea, additional domains of Trm10 homologues used to bind tRNAs are indicated in orange. The quality of the predictions can be assessed in **Supplementary Figure 2**.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 4.** Bacterial and eukaryotic RNAs Z bound to tRNA. (A) Crystal structure of RNase ZS from *B. subtilis* in complex with tRNA<sup>Thr</sup> (PDB 2FK6),<sup>35</sup> the RNase ZS dimer is represented with chain A in pink and chain B in purple. (B) CryoEM structures of human RNase Z<sup>L</sup> (ELAC2) in complex with pre-tRNA<sup>His</sup>-tRNA<sup>Ser</sup>(AGY) (PDB 8CBL, left),<sup>4</sup> and in complex with tRNA<sup>His</sup> containing the 3'-CCA sequence (PDB 9YE2, right)<sup>31</sup>. The CTD β-lactamase domain of ELAC2 is in pink, whereas the NTD is in purple.<sup>4</sup> For the sake of clarity, TRMT10C/SDR5C1 is not represented. The Zn<sup>2+</sup> ions are shown as grey spheres. (C) Structures of mt-pre-tRNA<sup>Tyr</sup> in complex with ELAC2 with TRMT10C/SDR5C1 (PDB 8RR4, green)<sup>32</sup> and without TRMT10C/SDR5C1 (PDB 8Z1G, blue).<sup>30</sup></figcaption></figure>

homodimer with one metallo-β-lactamase domain containing conserved motifs (HxHxDH, PxKxRN, AxDx) in each subunit crucial for tRNA cleavage (Figure 4a). RNase Z<sup>L</sup>, exclusive to eukaryotes, presumably arose from gene duplication and contains two metallo-β-lactamase domains (Figure 4b). Of these, only the C-terminal domain (CTD) retains enzymatic activity, while the NTD features an RNA-binding flexible arm or exosite,<sup>56</sup> resulting from a globular insertion in the β-lactamase fold.

In humans, RNase Z exists as two isoforms: ELAC1 (RNase Z<sup>S</sup>, 363 amino acids) and ELAC2 (RNase Z<sup>L</sup>, 826 amino acids). ELAC1, localized to the cytosol, repairs P-site tRNAs cleaved by ANGZF1 on stalled ribosomes.<sup>57</sup> It removes the 2',3'-cyclic phosphate to permit CCA re-addition by TRNT1.<sup>57</sup> Cells lacking ELAC1 specifically accumulate unrepaired tRNA intermediates upon the

induction of ribosome stalling.<sup>57</sup> ELAC2 has two alternative start codons to produce a nuclear and a mitochondrial-targeted forms, enabling it to process tRNAs in both compartments during their biogenesis.<sup>58,19,59</sup> This division of roles reflects evolutionary specialization, with ELAC1 and ELAC2 adapting to distinct subcellular functions. ELAC2 is sufficient for tRNA processing in the nucleus without other factors,<sup>60</sup> whereas *in vitro* studies have shown that ELAC2 requires the TRMT10C/SDR5C1 complex for efficient processing of most mt-tRNAs.<sup>11,32</sup>

Crystal structures of short-form RNase Z<sup>S</sup> have been solved in various organisms, including *Bacillus subtilis* (PDB 1Y44),<sup>61</sup> *Thermotoga maritima* (PDB 1WW1),<sup>62</sup> *Escherichia coli* (PDB 2CBN),<sup>63</sup> and humans (PDB 3ZWF). These studies revealed that RNase Z<sup>S</sup> enzymes function as dimers, with each subunit consisting of a metallo-
β-lactamase domain formed by a core of two oppos-
ing seven-stranded β-sheets flanked by three α-
helices in a βββαβαβαβ organization. The con-
served HxHxDH motif in each monomer coordi-
nates zinc ions essential for catalysis. The crystal
structure of *B. subtilis* RNase ZS bound to tRNA
(PDB 2FK6)³⁵ revealed that a single dimer can
simultaneously accommodate two tRNAs (Fig-
ure 4a). RNase ZS clamps onto the pre-tRNA
through interactions between the flexible arm of
one subunit and the elbow of the pre-tRNA, while
the helix α7 of the other subunit blocks the acceptor
arm (Figure 4a).

Long-form RNase Z, found in eukaryotes,
features two β-lactamase domains connected by a
long linker, as observed in the crystal structure of
*Saccharomyces cerevisiae* Trz1 (PDB 5MTZ)⁶⁴
and the cryoEM structures of ELAC2 (PDB 8CBL,
Figure 4b).⁴ Only the CTD of RNase Z⁻ retains the
conserved HxHxDH motif necessary for enzymatic
activity, while the NTD lacks this motif and does
not exhibit a metal-dependent hydrolase function.
The first structure of an RNase Z⁻ was solved for
the human RNA Z⁻ in complex with a mt-pre-tRNA
(PDB 8CBL).⁴ In this structure, ELAC2 is poised
to cleave the mitochondrial pre-tRNAHis-
tRNASer(AGY) pair of tRNAs.⁴ This structure has
revealed how ELAC2 processes structurally degen-
erate mitochondrial tRNAs (Figure 4b). The posi-
tively charged flexible arm of ELAC2 binds the T-
loop of pre-tRNAs, and on the other side, the CTD
helix of ELAC2 blocks the pre-tRNA. The 3'-trailer
is bound on a specific site on ELAC2 (Figure 4b).
TRMT10C interactions with ELAC2 are not required
for ELAC2 activity, but interactions between the
ELAC2 flexible arm and the T-loop are required.⁴
Residue D550, proposed to act as a base catalyst,
and residue H702, proposed to protonate the leav-
ing group,⁶⁵ are positioned near the scissile bond.

Several structures of different states of ELAC2
have since been published³⁰⁻³²: the apo form of
ELAC2 (PDB 8Z0P),³⁰ ELAC2 bound to pre-
tRNA**Tyr** without TRMT10C/SDR5C1 (PDB
8Z1G)³⁰ or with TRMT10C/SDR5C1 (PDB
8RR4),³² ELAC2 bound to pre-tRNA**Gln** with
TRMT10C/SDR5C1 (PDB 8RR3),³² ELAC2 bound
to pre-tRNA**His** with TRMT10C/SDR5C1 (PDB
9EY0 with WT ELAC2 and PDB 9EY1 with a cat-
alytically inactive mutant of ELAC2),³¹ ELAC2
bound to tRNA**His** with the 3'-CCA sequence (PDB
9EY2)³¹ and ELAC2 bound to a mature tRNA**Tyr**
(PDB 8Z1F).³⁰ They all confirm the recognition
mode of pre-tRNA by ELAC2.

For structures where the 3'-trailer is entirely
present (PDB 8CBL, 8RR3 and 8RR4), the 3'-
trailer is structured and is bound to a positively
charged region of ELAC2 at the exit of the active
site (Figure 4b, left), ⁴,³² Two Zn²⁺ ions are coordi-
nated by the motif ⁵⁴⁶HxHxDH⁵⁵¹, together with
H644, H702 and H724 of the ELAC2 CTD. The
CTD long helix acts as a hinge that adopts different

conformations depending on whether it guides the
pre-tRNA to the active site or helps release the
tRNA after 3'-trailer cleavage.³⁰ The 3'-CCA
sequence must remain intact after its addition by
TRNT1 to be aminoacylated and, therefore, must
not be cleaved by ELAC2. This is achieved by
excluding the 3'-CCA sequence from entering the
ELAC2 active site. Indeed, ELAC2 specifically rec-
ognizes the first two cytosines of the CCA sequence
with the ⁷²⁴HFSQRY⁷²⁹ motif, and an interaction
between residue Q92 of ELAC2 and the second
cytosine pulls the phosphodiester bond between
the two cytosines away from the catalytic site,
thereby preventing hydrolysis³¹ (Figure 4b, right).

ELAC2 also interacts with TRMT10C, at the level
of the tRNA elbow through hydrophobic interactions
between the ELAC2 flexible arm and the TRMT10C
NTD, and at the level of the ELAC2 CTH with the
methyltransferase domain of TRMT10C.⁴,³² For
mt-tRNA with a canonical structure, i.e., a well-
formed elbow, ELAC2 interacts directly with the
acceptor- and T-stems, and with the elbow region,
and interactions with TRMT10C are dispensable
for tRNA binding.³² Interestingly, two structures of
mt-pre-tRNA**Tyr** in complex with ELAC2 have been
solved, either with the pre-tRNA**Tyr** bound³² or
not³⁰ to the TRMT10C/SDR5C1 complex. The com-
parison of the pre-tRNA**Tyr** structures (Figure 4C)
shows that the TRMT10C interaction modifies the
conformation of the pre-tRNA**Tyr** anticodon loop,
but also the structure of the pre-tRNA**Tyr** elbow by
disrupting the interactions between the D- and T-
loops. This seems necessary for TRMT10C to
ensure the proper presentation of the T-loop of
mt-pre-tRNA to maturation enzymes.

Molecular basis of sequential pre-tRNA maturation in the nucleus

The molecular insights into human mitochondrial tRNA maturation provide valuable understanding of tRNA maturation processes in the nucleus. ELAC2 and TRNT1 also mature nuclear tRNAs. In contrast, nuclear and mitochondrial RNase P are evolutionarily and architecturally distinct in humans.⁶⁶,¹²,⁶⁷ Human nuclear RNase P is a large RNA-protein complex in which the RNA component catalyzes tRNA processing.⁶⁸ The cryoEM structure of nuclear RNase P bound to tRNA⁶⁷ shows that ELAC2 and nuclear RNase P cannot be simultaneously bound on the pre-tRNA. This imposes an order on the processing events, similar to mitochondrial tRNAs and factor exchange between the different enzymes. This order is strictly 5' first and then 3' because ELAC2 cannot bind a pre-tRNA with a 5'-leader sequence.⁴ The maturation events in the nucleus are thus depicted in Figure 5. Nuclear RNase P first binds to the pre-tRNA for 5'-leader cleavage, then it exchanges with ELAC2 for 3'-trailer processing, and lastly, ELAC2 exchanges with TRNT1 for 3'-CCA addition. Nuclear tRNAs
Human pre-tRNA maturation in the nucleus

<figure><img src="image_16.png" /><figcaption>**Figure 5.** Structural basis of human pre-tRNA maturation in the nucleus. AlphaFold3 prediction⁵⁴ of human nuclear pre-tRNA<sup>5</sup> is shown in red with the 5'-trailer and 3'-leader sequences in orange. Human nuclear RNase P structure (PDB 6AHU) is represented as a grey surface for the proteins, whereas the catalytic RNA is in light blue. ELAC2 from PDB 8CBL is represented as a marine surface, and TRNT1 (PDB 8CBM) as a tan surface.</figcaption></figure>

adopt a canonical L-shaped structure with an elbow
that is formed by interactions between the D- and
the T-loops. In this case, ELAC2 and TRNT1
directly bind the elbow of the nuclear pre-tRNAs.

Conclusion

In conclusion, TRMT10C/SDR5C1 facilitates the
processing of structurally degenerate mt-tRNAs
lacking the canonical elbow. While canonical
nuclear tRNAs and mt-tRNAs are recognized
through direct interactions with ELAC2, the
processing of non-canonical mt-tRNAs relies on
protein/protein interactions between ELAC2 and
TRMT10C. The results accumulated on mt-tRNAs
provide a molecular framework for understanding
tRNA maturation in both the nucleus and
mitochondria, highlighting the organelle-specific
need for additional factors. TRMT10C/SDR5C1
likely evolved as a dedicated mitochondrial tRNA
maturation platform to mitigate the structural
alterations of mt-tRNAs in bilaterian animals. By
pre-organizing the tRNA substrates, TRMT10C/
SDR5C1 may reduce the entropy cost during the
transition state of the enzymatic reaction, thus
enhancing the catalytic efficiency. This
compensatory role is likely not unique to
TRMT10C/SDR5C1. For example, in Arabidopsis
nuclei, the m<sup>2</sup>G46 tRNA-modification enzymes
TRM1A/TRM1B functionally cooperate with nuclear
RNase P during the early steps of cytosolic tRNA
biogenesis.⁶⁹ Future studies should explore this
compensatory function of tRNA modification
enzymes further, expanding our understanding of
their roles in tRNA maturation.

CRediT authorship contribution
statement

**Juhi Sikarwar:** Writing – review & editing, Writing – original draft. **Vincent Meynier:** Writing – original

draft. **Carine Tisné:** Writing – review & editing, Writing – original draft, Supervision, Conceptualization.

DECLARATION OF COMPETING INTEREST

The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to
influence the work reported in this paper.

Acknowledgements

The authors acknowledge financial support from
the European Union's Horizon 2020 research and
innovation programme under the Marie
Skłodowska-Curie grant agreement No
101034407 (FP-DYNAMO-PARIS COFUND
program), from the CNRS and the University
Paris Cite' (UMR8261), the Agence Nationale de
la Recherche (Project ARNTools ANR-19-CE07-
0028 and the Labex Dynamo ANR-11- LABX-
0011).

**Appendix A. Supplementary material**

Supplementary material to this article can be
found online at https://doi.org/10.1016/j.jmb.2025.
168989.

Received 15 December 2024;
Accepted 6 February 2025;
Available online 10 February 2025

Keywords:

TRMT10C;
ELAC2;
TRNT1;
mitochondria;
pre-tRNA processing
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