SARS-CoV-1 and SARS-CoV-2 proteome-wide virus-host protein interaction maps identified a putative interaction between catalytically inactive M^pro (Cys145Ala) and human TRMT1, but no such stable interaction was found using WT protease, suggesting M^pro may target and cleave TRMT1 in cells (Gordon et al., 2020b; Gordon et al., 2020a). A peptide sequence (527–534) located between the methyltransferase domain and zinc finger domain of human TRMT1 fits the cleavage consensus sequence of SARS-CoV-2 M^pro (Figure 1B). To determine if full-length (FL) TRMT1 is susceptible to cleavage by M^pro, we first measured proteolysis of human TRMT1 expressed and purified from E. coli by recombinant purified SARS-CoV-2 M^pro (Figure 2A). TRMT1 was incubated with either catalytically inactive M^pro Cys145Ala or active WT M^pro, and TRMT1 cleavage was monitored by western blot using two TRMT1-specific polyclonal antibodies: a dual-domain recognizing antibody specific for portions of both the methyltransferase and zinc finger domain (anti-TRMT1 460–659), and a single-domain recognizing antibody specific for only the zinc finger domain (anti-TRMT1 609–659). As expected, FL TRMT1 (~75 kDa) was stable during a 2 hr incubation with inactive Cys145Ala M^pro. However, when incubated with WT M^pro, the intensity of the band corresponding to FL TRMT1, as measured by both TRMT1 antibodies, was reduced by >90% after 2 hr. In western blots with cleaved TRMT1, the dual domain-recognizing anti-TRMT1(460–659) antibody showed the appearance of two new lower molecular weight bands, corresponding to the anticipated molecular weights of the M^pro-mediated TRMT1 cleavage products (~61 kDa TRMT1 methyltransferase domain and ~14 kDa TRMT1 zinc finger domain). After demonstrating that recombinant FL TRMT1 isolated from E. coli could be cleaved by SARS-CoV-2 M^pro, we next asked whether endogenous TRMT1 from human cells could be similarly cleaved by the viral protease. HEK293T cell lysate was incubated with either Cys145Ala or WT M^pro, and endogenous TRMT1 levels were monitored over time by western blot (Figure 2B). Incubation with Cys145Ala M^pro resulted in no change to the FL TRMT1 band over the course of 2 hr, while incubation with WT M^pro resulted in time-dependent proteolysis of FL TRMT1. Unlike in cleavage assays with recombinant TRMT1, no build-up of cleavage products was observed during M^pro-mediated proteolysis of endogenous TRMT1, suggesting instability and rapid degradation of cleaved TRMT1 fragments in human cell lysate. Together, these experiments demonstrate that both recombinant FL TRMT1 and endogenous FL TRMT1 in human cell lysate are viable substrates for cleavage by SARS-CoV-2 M^pro.

Next, we explored the consequences of M^pro-mediated cleavage of TRMT1 on both tRNA m2,2G modification and tRNA binding. TRMT1 was incubated overnight alone, with WT M^pro (to generate a fully cleaved TRMT1 product), or with M^pro Cys145Ala (no cleavage control). The M^pro cleavage reaction was monitored by western blot using anti-TRMT1 (460–659) antibody to confirm the depletion of FL TRMT1 by M^pro WT (Figure 2C and D, top). We then measured the impact of TRMT1 cleavage on its tRNA modifying activity by radiolabel-based methyltransferase assays with S-[methyl-¹⁴C]-adenosyl methionine (¹⁴C-SAM; Figure 2C, bottom) and its tRNA binding affinity by electrophoretic mobility shift assays (EMSAs) with FL human tRNA^phe (Figure 2D, bottom). Cleaved TRMT1 generated by incubation with M^pro WT exhibits a total loss of measurable tRNA methyltransferase activity, compared to TRMT1 incubated with M^pro Cys145Ala, which retains full activity comparable to TRMT1 alone (Figure 2C, Figure 2—source data 1). Similarly, we found that M^pro-cleaved TRMT1 also had reduced affinity for tRNA, whereas TRMT1 incubated with M^pro Cys145Ala had comparable tRNA binding affinity to TRMT1 alone (Figure 2D, Figure 2—source data 1). While cleaved TRMT1 still retained some affinity for tRNA, we observed in EMSA experiments that cleaved TRMT1-tRNA complexes migrate at significantly higher apparent molecular weights as compared to uncleaved TRMT1-tRNA complexes (Figure 2—figure supplement 1). This suggests that the TRMT1 cleavage products in vitro may form oligomers that have reduced affinity for tRNA, but based on the methyltransferase activity assays above, these oligomeric TRMT1-tRNA complexes cannot efficiently methylate bound tRNA. These observations are consistent with other studies that have shown truncated TRMT1 constructs lacking the zinc finger domain have reduced tRNA modification activity in cells (Dewe et al., 2017) or in vitro (Lu and Zhou, 2023). Together, our in vitro tRNA binding and modification assays directly show that M^pro-mediated cleavage of TRMT1 reduces its substrate tRNA binding affinity and completely incapacitates its tRNA m2,2G modification catalysis.

To visualize how TRMT1 is recognized and cleaved by the viral protease active site, we determined the co-crystal structure of catalytically inactive Cys145Ala mutant M^pro in complex with the TRMT1 peptide sequence corresponding to the expected cleavage site, human TRMT1 residues 526–536 (Figure 3, Figure 3—source data 1). The 1.9 Å resolution structure shows clear density for the TRMT1 peptide bound to the active site in one protomer of the M^pro dimer (Figure 3A, Figure 3—figure supplement 1A). As expected based on alignment with the M^pro consensus cleavage sequence, TRMT1 Gln530 occupies the critical S1 subsite pocket in the M^pro active site (Figure 3A), positioning the Gln530-Ala531 peptide bond directly adjacent to the His41/Cys145 catalytic dyad for proteolytic cleavage (Figure 3B). TRMT1 Leu529 corresponds to substrate position P2 and occupies the hydrophobic M^pro S2 pocket (Figure 3A), packing against M^pro residues His41, Met49, Met165, and Gln189 (Figure 3—figure supplement 1B). P3′ substrate residue TRMT1 Phe533 occupies the M^pro S3′ pocket (Figure 3A), which is composed by M^pro residues His41, Cys44, Met49, and the backbone of Thr45 (Figure 3—figure supplement 1C). TRMT1 peptide binding to M^pro is also mediated by numerous key sidechain and mainchain hydrogen bonding contacts (Figure 3C and D). The TRMT1 P1 Gln530 sidechain is specifically recognized by hydrogen bonding interactions with M^pro residues Phe140 (Figure 3C) and His163 (Figure 3D), consistent with previous structures of M^pro bound to viral peptide substrates (Shaqra et al., 2022). Additionally, the TRMT1 P2′ Asn532 sidechain interacts with M^pro Asn142 (Figure 3C). A number of TRMT1 peptide backbone atoms (from TRMT1 residues Arg528, Leu529, Gln530, Asn532, and Thr534) make hydrogen bond contacts with M^pro surface residues (M^pro Gly143, Glu166, and Gln189 contacts shown in Figure 3C; M^pro Thr24, Thr26, Cys145Ala, and His164 contacts shown in Figure 3D). The identified M^pro-targeted residues in human TRMT1 are conserved in the human population (i.e. no missense polymorphisms), showing that human TRMT1 can be recognized and cleaved by SARS-CoV-2 M^pro.

M^pro has more than 10 native viral polypeptide substrates with relatively high sequence variability outside the conserved P1 Gln and the adjacent P2 and P1′ residues. We compared our human TRMT1-bound M^pro structure with available SARS-CoV-2 M^pro structures bound to known viral polypeptide cleavage sequences (Figure 4A; nsp4/5, nsp5/6, nsp6/7, nsp8/9, nsp9/10, nsp10/11, and nsp15/16). The overall structures of M^pro are very similar for all peptide-bound structures, with M^pro backbone RMSD values all below 1.6 (Figure 4—figure supplement 1). Analysis of aligned peptide substrates in the M^pro active site shows that both TRMT1 and viral peptide backbones have nearly identical conformations for the N-terminal P4 → P1′ residues. In contrast, backbone geometries of C-terminal peptide residues (P2′ → P4′) diverge more significantly and fall into two distinct binding modes distinguished primarily by the P2′ Ψ dihedral angle (Figure 4B and C). The majority of viral peptide substrates adopt a binding conformation with P2′ Ψ≥157 ° in which the P3′ sidechain is positioned away from the M^pro surface (Figure 4D), which we have designated as the ‘P3′-out’ conformation. In contrast, TRMT1 and a single viral peptide substrate, nsp6/7, bind in a distinct conformation with P2′ Ψ≤116 ° (Figure 4C) in which the P3′ sidechain (Phe in TRMT1, Met in nsp6/7) is positioned toward the M^pro surface where it displaces M^pro Met49 to open and occupy pocket S3′ (Figure 4D). We have designated this uncommon binding mode as the ‘P3′-in’ conformation.

To further examine whether TRMT1 is a viable substrate for SARS-CoV-2 M^pro, we compared kinetic parameters for proteolysis of the TRMT1 (526–536) peptide with the nsp4/5 and nsp8/9 viral polypeptide cleavage sequences, using fluorogenic peptide cleavage assays. The TRMT1 peptide is cleaved noticeably slower than the nsp4/5 N-terminal auto-processing sequence (Figure 5A, Figure 5—source data 1), with an approximately 200-fold decrease in k_cat, 4-fold decrease in K_M, and 50-fold decrease in catalytic efficiency (k_cat/K_M) compared to M^pro-mediated cleavage of the nsp4/5 peptide (Figure 5B, Figure 5—source data 1). However, TRMT1 is cleaved with very comparable kinetics to the viral nsp8/9 cleavage sequence, with k_cat, K_M, and k_cat/K_M values all within ~3-fold for TRMT1 vs nsp8/9 peptide cleavage. These kinetic experiments show that, while the TRMT1 M^pro target sequence is cleaved less efficiently than the canonical nsp4/5 auto-processing site, the TRMT1 sequence is cleaved with kinetics very similar to the known nsp8/9 M^pro cleavage site in the viral polypeptide. This suggests that TRMT1 (526–536) is a viable substrate for M^pro-targeted proteolysis.

We next compared the M^pro-peptide interactions observed in TRMT1-, nsp4/5-, and nsp8/9-bound structures and identified several M^pro residues involved in direct substrate contacts that differ between the TRMT1- and nsp4/5- or nsp8/9-bound structures (Figure 5C). In the TRMT1-bound structure, M^pro Met49 is shifted to accommodate TRMT1 P3′ substrate residue Phe533 binding in the S3′ pocket, and the M^pro Asn142 and Gln189 sidechains are repositioned to form hydrogen bonds with the TRMT1 peptide backbone. To test how these M^pro active site residues affect TRMT1 recognition and catalysis, we generated single alanine point mutations at each of these three sites and measured kinetic parameters for M^pro-mediated proteolysis of nsp4/5 and TRMT1 peptides. Additionally, we mutated the P1′ TRMT1 Ala531 to Ser, the P1′ residue found in nsp4/5 and nearly all of the other M^pro-targeted viral cleavage sequences, to test the effects of mutating a key position in the substrate peptide. Surprisingly, we saw no substantial changes in proteolysis kinetics for cleavage of either nsp4/5 or TRMT1 substrates with any of the tested M^pro mutants (Met49Ala, Asn142Ala, Gln189Ala) or the TRMT1 Ala531Ser mutant peptide (Figure 5D, Figure 5—source data 1, Figure 5—figure supplement 1), suggesting none of these residues alone play a significant role in determining differential cleavage rates for TRMT1 vs nsp4/5.

To determine whether the TRMT1 sequence at the M^pro cleavage site is unique to human TRMT1 and whether TRMT1 bears signatures of molecular arms-races with pathogens during mammalian evolution, we performed phylogenetic and positive selection analyses (Tenthorey et al., 2022; Sironi et al., 2015). Through an evolutionary screen, Cariou et al., 2022, previously identified some signatures of rapid evolution in primate TRMT1, potentially driven by adaptation to ancient viral pathogens, including coronaviruses. Through comprehensive analyses, we identified rapidly evolving sites at the N- and C-termini of primate TRMT1, but we did not find any evidence of sites under positive selection at the M^pro-mediated TRMT1 (526–536) cleavage sequence (Figure 6—figure supplement 1A, Figure 6—source data 1). In fact, this sequence is highly conserved in primates (Figure 6A). A TRMT1 sequence analysis at the mammalian level further showed that the M^pro cleavage site of TRMT1 (residues 526–536) is highly conserved in most mammals, including in bats that are the reservoir of SARS-CoVs (Figure 6B). However, one exception is in rodents, where there has been a Q to K substitution fixed in all Muroidea (mouse, rat, hamster, etc.) (Figure 6C, Figure 6—figure supplement 1B). To test the impact of this substitution on M^pro-mediated cleavage, we generated a TRMT1 Gln530Lys mutant and incubated it with M^pro WT for a period of 2 hr (Figure 6D). We found that TRMT1 Gln530Lys was resistant to M^pro cleavage and did not observe the formation of cleavage products (e.g. TRMT1 MT domain) compared to cleavage of TRMT1 WT under the same proteolytic conditions. This is consistent with a more recent report showing that mouse or hamster TRMT1 is not cleaved by M^pro when these enzymes are co-expressed in cells (Lu and Zhou, 2023). Our results confirm that the TRMT1 Gln530Lys substitution at the invariant P1 glutamine residue is sufficient to completely prevent TRMT1 proteolysis in vitro, strongly suggesting this mutation would prevent SARS-CoV M^pro-directed TRMT1 cleavage during Muroidea infection.

To further support the structural and biochemical data described above and attempt to understand how peptide substrate binding geometry may or may not be linked to M^pro-catalyzed cleavage efficiency, we carried out MD simulations using nsp4/5-, nsp8/9-, and TRMT1-bound M^pro complexes. MD simulations show TRMT1 is stably bound to the M^pro active site and primarily adopts the P3′-in conformation observed in the crystal structure, where TRMT1 Phe533 occupies the S3′ pocket with a probability of ~65% over the course of the 100 ns simulation (Figure 7A). In contrast, simulations of the nsp4/5-M^pro complex show the nsp4/5 peptide primarily adopts the P3′-out conformation, where nsp4/5 Phe531 is oriented away from the M^pro surface with ~70% probability, consistent with the observed nsp4/5 binding mode in the crystal structure. This computational analysis shows that while P3′-in and -out conformations can interconvert, the dominant binding modes for TRMT1 and nsp4/5 peptide substrates (P3′-in vs -out, respectively) are consistent across the crystal structures and MD simulations. Furthermore, because these conformations differ primarily at the C-terminal end of the peptide but have very similar geometries at the scissile amide bond, we find that the computationally and experimentally determined substrate binding poses fail to explain the large differences in cleavage kinetics observed for TRMT1 vs nsp4/5.

Previous structural and geometric analysis of diverse serine protease substrate-inhibitor complexes has shown that the consensus nucleophilic attack angle of the catalytic residue in the reactive Michaelis complex is approximately 90° (Radisky, 2002). We next asked whether differences in nucleophilic attack angle for the catalytic M^pro Cys145 measured over the course of the MD simulation might be able to explain changes in M^pro-mediated cleavage kinetics. The distribution of the S–C=O nucleophilic attack angle shows that the M^pro-nsp4/5 complex has only ~9% increased probability to fall within 89±7°, as compared with the M^pro-TRMT1 complex over the course of the simulation (Figure 7B). Thus, while the nsp4/5 peptide shows slightly more favorable positioning of Cys145 for nucleophilic attack in the Michaelis complex during the first step of cleavage, this is not nearly sufficient to explain the large difference (~200-fold in k_cat) in experimentally observed cleavage kinetics between nsp4/5 and TRMT1. Taken together with our TRMT1-M^pro structure and kinetic analysis of M^pro and TRMT1 mutants, these results strongly suggest that kinetic discrimination between peptide cleavage rates is likely to occur during a later step in the M^pro-catalyzed cleavage reaction that follows substrate binding, Michaelis complex formation, and initial nucleophilic attack, similar to some serine proteases (Radisky, 2002).

M^pro constructs used for biochemical and kinetic studies were subcloned into a pETARA expression vector containing N-terminal GST and C-terminal His-tags using Gibson assembly. Human codon optimized WT M^pro was obtained from Addgene (catalog #141370). M^pro single-point mutants (M49A, N142A, Q189A) were introduced using site-directed mutagenesis by whole plasmid PCR. Each construct contained the M^pro autocleavage sequence (AVLQ) after the N-terminal GST tag to allow for self-cleavage and a native M^pro N-terminus. Constructs were transformed into E. coli Rosetta(DE3)pLysS cells and plated on LB agar plates with 50 µg/mL ampicillin. Overnight seed cultures were grown from single colonies in LB media with 50 µg/mL ampicillin, at 37°C with shaking at 200 rpm. A 1:100 dilution of seed culture to 1 L LB media was prepared and grown at 37°C with shaking at 200 rpm. After an OD₆₀₀ of 0.6 was reached, cells were induced with 1 mM IPTG and incubated overnight at 18°C with shaking. Cultures were centrifuged at 7500 × g and the supernatant was discarded. Harvested cells were sonicated in lysis buffer (25 mM Tris, 300 mM NaCl, 10 mM imidazole pH 8.0), centrifuged at 14,500 × g for 45 min, and recovered, clarified lysate was loaded onto 1.5 mL (bed volume) equilibrated Thermo Scientific HisPur Ni-NTA Resin for 30 min with gentle mixing. Resin was added to a standard gravity column and washed with two 25 mL washes of lysis buffer and two 25 mL washes with lysis buffer+25 mM imidazole, and eluted with 10 mL lysis buffer+250 mM imidazole. Eluate was concentrated to ~2 mL using a using 10 kDa MWCO centrifugal concentrator and applied to a Cytiva HiLoad 16/600 Superdex 200 pg column for size exclusion chromatography. M^pro-containing fractions were concentrated to between 10 and 25 mg/mL, flash-frozen with liquid nitrogen, and stored at –70°C in 50 mM Tris, 1 mM EDTA, 2 mM DTT, pH 7.3.

Catalytically inactive M^pro C145A mutants used for protein crystallography experiments were prepared by subcloning M^pro C145A from Addgene (catalog #141371) into a pET28a expression vector containing an N-terminal His-tag and tobacco etch virus (TEV) protease cleavage site (ENLYFQGS). The M^pro C145A construct was transformed and expressed in E. coli as described above. Cultures were centrifuged at 7500 × g and the supernatant was discarded. Harvested pellets were sonicated in lysis buffer (25 mM Tris, 300 mM NaCl, pH 8.0) and clarified the lysate by centrifugation at 14,500 × g. Recovered lysate was loaded onto 3.75 mL (bed volume) equilibrated Thermo Scientific HisPur Ni-NTA Resin for 30 min with gentle mixing. Resin was added to a standard gravity column and washed with two 25 mL washes of lysis buffer and two 25 mL washes with lysis buffer+25 mM imidazole, and eluted with lysis buffer+300 mM imidazole. Eluate was incubated overnight at room temperature with recombinant purified TEV protease at a 1:50 molar ratio to cleave the N-terminal His-tag. An Ni-NTA backpass was performed and the flow-through was concentrated, buffer exchanged into 25 mM Tris, 25 mM NaCl, pH 7.5 using a using 10 kDa MWCO centrifugal concentrator, and applied to a Cytiva HiTrap 5 mL Q HP column. Protein was eluted with a gradient of 25 mM to 1 M NaCl and M^pro-containing fractions were pooled, concentrated, and applied to a Cytiva HiLoad 16/600 Superdex 200 pg size exclusion chromatography column and eluted with an isocratic gradient using 25 mM Tris, 25 mM NaCl, pH 7.5. The protein-containing fractions were concentrated to approximately 26 mg/mL, flash-frozen with 10% glycerol in liquid nitrogen, and stored at –70°C.

The human TRMT1 gene sequence (NM_001136035.4) with a 1–18 amino acid N-terminal deletion (to remove the mitochondrial targeting peptide) was synthesized and subcloned by GenScript into pET22b(+) with an N-terminal pelB leader sequence and a C-terminal His-tag. The TRMT1 construct was transformed into E. coli Rosetta(DE3)pLysS cells as described above, and induced using 0.2 mM IPTG. Harvested cells were sonicated in lysis buffer (20 mM Tris-HCl, pH 8.5, 500 mM NaCl, 5 mM imidazole, 10% glycerol, 5 mM BME, cOmplete EDTA-free Protease Inhibitor Cocktail), clarified at 14,500 × g, and recovered lysate was loaded onto a pre-equilibrated Cytiva 5 mL HisTrap column. The loaded HisTrap column washed with lysis buffer+20 mM imidazole, and protein was eluted using a gradient elution with lysis buffer+250 mM imidazole. Fractions containing TRMT1 were pooled, concentrated, and loaded onto a Cytiva HiLoad 16/600 Superdex 200 pg size exclusion chromatography column and eluted with an isocratic gradient using 20 mM Tris pH 8.5, 500 mM NaCl, 2 mM DTT. The protein-containing fractions were concentrated to approximately 30 mg/mL, flash-frozen in liquid nitrogen, and stored at –70°C.

Purified M^pro C145A was incubated at room temperature for 1 hr with TRMT1 (526–536) peptide (EPRLQANFTIR, synthetic peptide obtained from Peptide 2.0) in 25 mM Tris, 25 mM NaCl, pH 7.4 at a 1:3 molar ratio with the final concentrations of 7 mg/mL M^pro C145A and 619 µM TRMT1 (526–536). The M^pro and TRMT1 solution was mixed 1:1 with well solutions consisting of 20–21.5% PEG 3350 and 100 mM NaCl in 24-well hanging drop VDX plates with a final drop volume of 1 µL. Initial crystals were harvested, crushed, and seeded into new hanging drops, with components as listed above, using cat whiskers. Single crystals were harvested and flash-frozen in liquid nitrogen with 20% glycerol in well solution as a cryoprotectant. Diffraction data were collected at the National Synchrotron Light Source II (NSLS II) Highly Automated Macromolecular Crystallography (AMX) (Schneider et al., 2022) beamline 17-ID-1 at the Brookhaven National Laboratory on an Eiger 9M Pixel detector at 100 K and a wavelength of 0.920219 Å. Diffraction data were indexed, integrated, and scaled using XDS. The M^pro-TRMT1 structure was solved in space group P2₁2₁2₁ using the Phaser package in the CCP4 suite and a modified PDB 7MGS as search model. After initial rounds of refinement in PHENIX to model M^pro residues, the TRMT1 peptide residues 526–534 were manually built into the F_o-F_c map using COOT. Subsequent rounds of automated refinement and water placement using PHENIX and manual adjustments including modeling sodium ions and glycerol molecules in COOT were used to obtain the final structure.

Fluorogenic assays measuring kinetic parameters for cleavage of peptide substrates were carried out in triplicate in Corning Low Volume 384-well Black Flat Bottom Polystyrene NBS Microplates following a similar procedure as Lee et al., 2020. Peptide cleavage reactions were carried out at 50 nM M^pro enzyme in 50 mM Tris pH 7.3, 1 mM EDTA, 2 mM DTT, and 20% DMSO, with quenched fluorescent peptide substrates ranging in concentration from 0.097 to 100 µM. Synthetic peptide substrates were obtained from Peptide 2.0: TRMT1 (MCA-EPRLQANFTIR-K(Dnp)K), nsp4/5 (MCA-SAVLQSGFRKM-K(Dnp)K), and nsp8/9 (MCA-AVKLQNNELSP- K(Dnp)K) where MCA = 7-methoxycoumarin-4-acetic acid and Dnp = dinitrophenyl. Using a Tecan Spark microplate reader, the fluorescence intensity was monitored every 10 s over a 3 min time course, with excitation at 320 nm and emission at 405 nm. A calibration curve of MCA-AVLQ product fluorescence intensities from 12 to 0.006 µM was measured and used to generate an RFU to µM conversion factor. A correction for the inner filter effect (IFE_corr) was determined using the formula IFE_corr = [fluorescence_{MCA_product+peptide} – fluorescence_peptide]/fluorescence_MCA where fluorescence_{MCA_product+peptide} = the fluorescence of MCA-AVLQ mixed with quenched peptide substrate, fluorescence_peptide = the fluorescence of the peptide alone, fluorescence_MCA = fluorescence of MCA-AVLQ product alone (Figure 5—source data 2; Liu et al., 1999). Plots of initial rate (µM/min) vs peptide substrate concentration were fit to the Michaelis-Menten equation to determine V_max and K_M kinetic parameters for M^pro-mediated peptide cleavage using Origin 2021 software.

HEK293T cells used in this study were obtained from ATCC with confirmed identity by STR profiling and no detection of mycoplasma, bacterial, or fungal contamination.

Freshly passaged HEK293T cells were incubated at 37°C with 5% CO₂ in growth media (DMEM media with 10% FBS and penicillin [100 U/mL]/streptomycin (100 µg/mL)). Growth media was replaced every 3 days, and cells were trypsinized and passaged approximately every 7 days, when cells reached 80–90% confluency. To passage, growth media was aspirated, and cells were gently washed with prewarmed PBS. PBS was then aspirated and a 1:2.5 mixture of 0.25% Trypsin in HBSS with 0.2 g/L EDTA to PBS was added to adherent cells and incubated at 37°C for 2 min to disaggregate. A 3× dilution with growth media was used to inactivate trypsin. The cells were gently mixed to attain a homogenous suspension and diluted 1:20 with growth media and added into a new cell culture plate. For lysis, cells were trypsinized as described above, and the resulting cell suspension was spun down at 1250 rpm for 3 min. Cell pellets were washed in ice-cold PBS three times, and spun at 1250 rpm for 3 min, aspirating PBS in between each wash. Lysis was performed on a PBS suspension of cells by performing 3 consecutive freeze/thaw cycles by flash-freezing in liquid nitrogen and thawing at 37°C. Lysate was collected after centrifugation at 13,000 rpm for 10 min and stored at –70°C.

TRMT1 proteolysis reactions with recombinant TRMT1 isolated from E. coli were performed with 10 µM WT or C145A M^pro in 20 mM HEPES, 105 mM NaCl, 20% glycerol, pH 7.0. Recombinant FL TRMT1 from E. coli was diluted sevenfold. Reaction was incubated at 37°C and timepoints were quenched by adding to SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 1.0% [wt/vol] SDS, 10% [vol/vol] glycerol, 0.1% [wt/vol] bromophenol blue, 0.1 M DTT) and boiled for 5 min. TRMT1 levels and fragment sizes at different reaction timepoints were assessed by western blot.

TRMT1 proteolysis assays using endogenous human TRMT1 from HEK293T lysate were carried out using cell lysate preincubated with 1 mM phenylmethylsulfonyl fluoride prior to reaction to prevent proteolysis by mammalian-specific proteases. M^pro proteolysis reactions were performed with 10 µM WT or C145A M^pro in 25 mM Tris, 25 mM NaCl, 20% glycerol, pH 7.5. Reaction was incubated at 37°C and timepoints were quenched by adding to SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 1.0% [wt/vol] SDS, 10% [vol/vol] glycerol, 0.1% [wt/vol] bromophenol blue, 0.1 M DTT) and boiled for 5 min. TRMT1 levels and fragment sizes at different reaction timepoints were assessed by western blot.

Quenched samples from the proteolysis assays were loaded (10 µL) onto Bio-Rad TGX 4–15% polyacrylamide gels and run for 30 min at 180 V. Gels were blotted onto PVDF membranes using a Bio-Rad Trans blot Turbo for 7 min at 2.5 A. The blot was incubated in blocking solution (5% non-fat milk in 1× Tris-Buffer Saline with 0.1% Tween) at room temperature for 1 hr. All antibodies were diluted in blocking solution. TRMT1-specific antibodies corresponding to amino acid regions 460–659 (Invitrogen Rabbit Anti-TRMT1 [ref: PA5-96585]) and 609–659 (Bethyl Laboratories Rabbit Anti-TRMT1 [ref: A304-205A]) and housekeeping protein GAPDH (Invitrogen Rabbit Anti-GAPDH [ref: PA-1987]) were utilized for primary antibody staining at 1:2,000 dilution for TRMT1 antibodies and 1:100,000 for GAPDH antibodies, and were stained by overnight incubation at 4°C. Extensive washing of blot with 1× Tris-buffered saline with 0.1% Tween were performed after primary antibody staining. Invitrogen Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (ref: A16096) was used for secondary staining at a dilution of 1:10,000 for 1 hr at room temperature. Extensive washing of blot with 1× Tris-buffered saline with 0.1% Tween were performed after secondary antibody staining. Clarity Western ECL Substrate (ref: 1705060) was added to blot and incubated at room temperature for 5 min. Western blots were visualized on a Protein Simple FluorChem R imager.

A double-stranded template of tRNA^phe (Homo sapiens Phe-GAA, sequence with T7 promoter: TAATACGACTCACTATAGCCGAAATAGCTCAGTTGGGAGAGCGTTAGACTGAAGATCTAAAGGtCCCTGGTTCGATCCCGGGTTTCGGCA) was used at a concentration of 150 nM in the transcription mixture of 0.05% Triton X-100, 5 mM DTT, 5 mM rNTPs, 0.1 mg/mL RNA polymerase, 2 U/mL TIPP, 40 mM Tris-HCl, pH 7.5, 50 mM MgCl₂, and 2 mM spermidine. The reaction was incubated at 37°C for 3 hr, then treated with DNase RQ1 and incubated at 37°C for 1 hr. The reaction was quenched with 50 mM EDTA and 50% formamide and heated for 5 min at 95°C. The quenched material was loaded onto a large-scale 8% gel PAGE denaturing UREA gel and run at 50 mA for ~2.5 hr. Using a handheld UV lamp and a silica plate, the tRNA band was excised, shred, and nutated overnight at room temperature in 0.6 M NaOAc, pH 6.0, 1 mM EDTA, and 0.01% SDS. The solution was filtered and extracted using phenol:chloroform:IAA, 25:24:1, pH 6.6, centrifuging for 5 min at 1500 × g and collecting the aqueous phase, then precipitating with 80% ethanol overnight at –20°C. The tRNA pellet was air-dried at room temperature and brought up in molecular grade water. tRNA was then re-folded by heating at 80°C for 2 min, 60°C for 2 min, and then adding 10 mM MgCl₂ and cooling on ice for 30 min. The final material was flash-frozen at ~150 μM and stored at –80°C.

Overnight M^pro cleavage reactions were prepared with 0.5 μM TRMT1 in 50 mM Tris pH 8.0, 50 mM MgCl₂, 100 µg/µL BSA, 2 mM DTT and added to 20 μM M^pro WT, 20 μM M^pro C145A, or no protease. Timepoints at 0 and 18 hr were taken for each protease condition and assessed for reaction completion by western blot. The completed M^pro WT, M^pro C154A, or no protease (mock) cleavage reactions were used directly in methyltransferase activity assays with 0.1 μM final concentration of TRMT1, 5 μM tRNA^phe, and 30 μM ¹⁴C SAM (4.2 mCi/mmol) in 50 mM Tris pH 8.0, 50 mM MgCl₂, 100 µg/µL BSA, 2 mM DTT. Reactions were incubated at 37°C and 5 μL timepoints were taken at 0, 0.5, 2, and 4 hr and processed using Zymo RNA Clean & Concentrator kits to isolate tRNA from the reaction mixture. tRNA was eluted in 15 μL water and added to 10 mL of Ultima Gold Scintillation Fluid (ref: 6013329). Counts per minute (CPM) were measured using Quantulus Scintillation Counter CH3 channel (lower level 0, upper level 156, 1 min count time). A standard curve using known concentrations and specific activity of ¹⁴C-SAM was produced to convert CPM to concentration of labeled product. Graphs were generated using Origin2023b and fit using a Michaelis-Menten equation.

Overnight M^pro cleavage reactions were prepared with 14 μM TRMT1 in 20 mM Tris-HCl, pH 8.5, 300 mM NaCl, 2 mM DTT, 20% glycerol and added to 140 μM M^pro WT, 140 μM M^pro C145A, or no protease. Timepoints at 0 and 18 hr were taken for each protease condition and assessed for reaction completion by western blot. The completed M^pro WT, M^pro C154A, or no protease (mock) cleavage reactions were used directly to generate a dilution series of TRMT1 and each dilution was mixed with tRNA^phe (final TRMT1 concentrations ranged from 7.78 to 0.13 μM; final tRNA^Phe concentration was 0.2 μM). The TRMT1-tRNA binding reactions were incubated for 1 hr at room temperature, 8 μL of each reaction was loaded onto a 5% TBE non-denaturing gel, and run at 50–60 V for approximately 2.5 hr at 4°C. Each gel was stained with 1× SYBR Gold in 0.5× TBE for 30 min at room temperature and then imaged using a Protein Simple FluorChemQ imager. Band intensities for bound and unbound tRNA were quantified using ImageJ software (Schneider et al., 2012) and used to calculate fraction bound values at each TRMT1 concentration. Graphs were generated using Origin2023b and fit using a Hill plot.

Extensive MD simulations in explicit water have been carried out to investigate M^pro-nsp4/5 and M^pro-TRMT1 complexes starting from their corresponding crystal structures (PDB id: 7MGS [MacDonald et al., 2021] and 8D35), respectively. In original crystal structures, the catalytic Cys145 has been mutated to Ala, M^pro-nsp4/5 is in the monomer form, while the M^pro-TRMT1 complex is in the dimer form. Since the active form of M^pro should be dimer (Zhang et al., 2020b), the structure of the M^pro-nsp4/5 (PDB id: 7MGS) was superimposed to the M^pro-TRMT1 (PDB id: 8D35) to model the dimer form of M^pro-nsp4/5, and the catalytic Cys145 in both complexes has been modeled using ‘swapaa’ command in ChimeraX (Pettersen et al., 2021). The original crystal water molecules and ions within 5 Å of the protein-substrate complex are kept. PDB2PQR (Jurrus et al., 2018) was employed to add hydrogen atoms to both complexes at pH 7. Subsequently, the His41 was manually changed to Hip41 (positively charged) by adding an additional hydrogen atom to the imidazole ring in ChimeraX, and Cys145 was manually changed to Cym (negatively charged) by removing the hydrogen atom on the thiol group in ChimeraX. Each system was neutralized by adding counterions for Na⁺ and Cl^-, and solvated in a rectangular water box of TIP3P water molecules with 12 Å buffer.

For each prepared simulation system, 3000 steps of steepest descent plus 2000 steps of conjugate minimization was performed with harmonic restraints using a force constant of 20 kcal/mol/Ų applied to all heavy atoms coming from the crystal structure, and then the whole system was minimized without restraints by 7000 steps of steepest descent and 3000 steps of conjugate gradient minimization. After minimization, three independent equilibration and MD replicas were carried out for 100 ns with the same starting structural configuration but different initial velocities. The equilibration was conducted in five steps: (a) 50 ps constant volume ensemble (NVT) MD simulation with 10 kcal/mol/Ų restraints on all heavy atoms from the crystal, and the whole system was heated from 10 to 300 K gradually. (b) 50 ps isothermal isobaric ensemble (NPT) MD with 10 kcal/mol/Ų restraints on all heavy atoms from the crystal at 300 K. (c) 200 ps NPT MD with 5 kcal/mol/Ų restraints on all heavy atoms from the crystal at 300 K. (d) 200 ps NPT MD with 2 kcal/mol/Ų restraints on all heavy atoms from the crystal at 300 K. (e) 200 ps NPT MD with 1 kcal/mol/Ų restraints on all heavy atoms from the crystal at 300 K. Finally, production MD simulations were carried out for 100 ns at a constant temperature of 300 K and a constant pressure of 1 atm. Langevin thermostat and Berendsen barostat methods were employed to maintain the temperature and pressure, respectively. The atomic coordinates of the complexes were saved every 1 ps to obtain the trajectories for analysis.

All MD simulations were conducted with Amber 20 (Case, 2020) package using Amber FF 14SB force field (Maier et al., 2015). AmberTools (Case, 2020) was utilized for preparing topology and coordinate files for the simulated systems cutoff of 10 Å was set for calculating van der Waals interactions, and the particle mesh Ewald (Darden et al., 1993) method with a cutoff of 10 Å was employed to treat electrostatic interactions. The SHAKE algorithm (Andersen, 1983) was used to constrain covalent bonds to allow the integration time step of 2 fs.

For the mammalian-wide sequence analyses, the TRMT1 amino acid alignment was retrieved with OrthoMaM (Orthologous Mammalian Markers) v10c (Scornavacca et al., 2019), using human TRMT1 (ENSG00000104907) as query. The cleavage site was located and the sequence logo of the 526–536 region was generated using WebLogo3 (https://weblogo.threeplusone.com/).

For the primate phylogenetic analyses, TRMT1 orthologous sequences were retrieved using the DGINN pipeline with the human TRMT1 CCDS12293 as query (Picard et al., 2020). TRMT1 sequences from additional primate species were retrieved using NCBI Blastn (Figure 6—source data 1).

For the rodent phylogenetic analyses, the rodentia orthologous protein and mRNA reference sequences to the human TRMT1 (ENSG00000104907) gene were collected from OrthoMaM and ncbi HomoloGene (https://www.ncbi.nlm.nih.gov/homologene; Figure 6—source data 1). Because most rodent species lack a reference CDS, we used BLOSUM62 matrix implemented in GeneWise from EMBL-EBI tools (Birney et al., 2004; Madeira et al., 2019), to identify the TRMT1 ORFs. Codon alignments of primate and rodent TRMT1s were performed using WebPrank (Löytynoja and Goldman, 2010) with default settings for primates (trust insertions + F, gap rate = 0.05, gap length = 5, K=2) and with a gap rate of 0.1 for rodents. Positive selection analyses on the codon alignments were performed with HYPHY/Datamonkey (Kosakovsky Pond et al., 2020; Weaver et al., 2018), using two branch-site models, BUSTED (version 3.1)(Murrell et al., 2015) and aBSREL (version 2.2) (Smith et al., 2015), and two site-specific models, MEME (version 2.1.2) (Murrell et al., 2012) and FUBAR (version 2.2) (Murrell et al., 2013).

To identify missense polymorphisms or variants in TRMT1 at minor allele frequency above 0.005 in human population, we mined the dbSNP database https://www.ncbi.nlm.nih.gov/snp/.

All materials, including plasmids generated in this study, are available from the authors upon request.