A disease-causing isoleucyl-tRNA synthetase variant leads to altered protein complex formation and cellular stress response

TL;DR

A mutation in an enzyme involved in protein synthesis causes cellular stress and reduced protein levels, possibly leading to disease.

Contribution

The study reveals a new disease mechanism involving altered protein complex formation and stress response, not directly linked to enzyme activity.

Findings

A disease-causing mutation in IARS1 reduces protein levels and affects complex formation.

The mutation alters the integrated stress response pathway, especially under low-glucose conditions.

Catalytic activity remains intact, but protein stability and complex formation are disrupted.

Abstract

Aminoacyl-tRNA synthetases are key enzymes in protein synthesis, as they catalyze the attachment of amino acids to their designated, cognate tRNAs. As such, mutations in aminoacyl-tRNA synthetases are associated with severe diseases, such as neurodevelopmental disorders. Many of these mutations occur in the catalytically active site or tRNA-binding domains; however, others can affect domains associated with multisynthetase complex formation. Here, we investigate a disease-causing mutation in the unique-I domain of isoleucyl-tRNA synthetase (IARS1, IleRS), which mediates IleRS interactions within the multisynthetase complex. Interestingly, levels of the resulting protein were severely reduced in comparison to wt IleRS. While bulk protein synthesis and cell proliferation were not affected, the integrated stress response signaling pathway was altered. This change was exacerbated in low-glucose medium, suggesting that mutant cells could respond differently to cellular stress. Our study hints at a possible underlying disease mechanism, where catalytic activity might not be affected but instead complex formation and protein stability.

Figures

Full text

Aminoacyl-tRNA synthetases (aaRSs) are a group of enzymes responsible for linking tRNAs with amino acids through esterification reactions to provide substrates for protein synthesis (1, 2). aaRSs are essential for all life forms as interpreters of the genetic code by recognizing both their corresponding amino acid and the cognate tRNA (3, 4). Charged tRNAs are then used by the ribosome for protein synthesis, where base pairing of the tRNA anticodon to mRNA determines the sequence of amino acids in a protein. aaRSs are named after the amino acid they recognize, for example, isoleucyl-tRNA synthetase (IleRS, IUPAC gene name: IARS1) charges isoleucine on tRNA.

While aaRSs are generally active as either monomers or dimers, in multicellular organisms, certain aaRSs form a protein complex called the multisynthetase complex (MSC) (5). The composition and complexity of the MSC vary across species (6, 7). In mammals, the MSC includes eight aaRSs with nine enzymatic activities (ArgRS/RARS1, GlnRS/QARS1, MetRS/MARS1, GluProRS/EPRS, IleRS/IARS1, LeuRS/LARS1, AspRS/DARS1, and LysRS/KARS1) (8). The MSC also contains three adaptor proteins, AIMP1, 2, and 3, which are essential for complex integrity (9, 10, 11, 12, 13). The individual proteins contain domains that facilitate the intermolecular interactions within the MSC (14), including leucine zippers (15) and glutathione-S-transferase-like motifs (16). In addition, individual aaRSs can contain so-called unique (UNE) domains, which can be involved in complex formation: The UNE-L domain in LeuRS (17) and the two UNE-I domains in IleRS mediate their integration into the MSC (18). Unlike other enzymes, where complex formation is a prerequisite for function, aaRSs are enzymatically active both within and outside the MSC (19). The function of the MSC is therefore still ambiguous (20), as exclusion of individual aaRSs does not necessarily affect global protein synthesis (21), suggesting other roles in regulating noncanonical functions or specialized translation.

In addition to their contributions to protein synthesis, aaRSs also mediate diverse cellular processes through noncanonical functions. These include but are not limited to the regulation of mRNA transcription (22) and translation (23, 24, 25, 26, 27) as well as cell signaling (28, 29, 30). Of note, LeuRS controls the mechanistic target of rapamycin (mTOR) pathway in response to glucose levels (30, 31), and IleRS is associated with BRCA and PI3K signaling (32, 33).

Given their central role in protein synthesis, mutations in aaRSs can cause severe disorders in humans (34). These disorders frequently involve multiple organs, including the central nervous system, and feature leukodystrophies (35). While many of these disease-causing mutations are located in the catalytic domain or affect tRNA binding, individual variants fall outside domains classically associated with aaRSs' enzymatic activity (36). The most common RARS1 mutation in patients with hypomyelinating leukodystrophy leads to the preferential expression of a variant lacking the leucine zipper, a domain that anchors ArgRS to the MSC (37, 38). In a model cell line, decoding of arginine codons was not diminished, and overall bulk protein synthesis as well as cell viability were comparable to control cells (21). Similarly, disease-associated point mutations have been found in the two UNE-I domains of IleRS (35), both of which are noncatalytic domains that mediate IleRS MSC integration (14, 33, 39).

We sought to investigate the mechanism underlying the pathogenicity of the IleRS variant c.3521T>A, I1174I>N (35), hereafter referred to as I1174N. Since this mutation resides in the noncatalytic UNE-I domain, interference with aminoacylation activity alone seemed unlikely. We therefore hypothesized that I1174N could alter the interaction of IleRS with other proteins, including components of the MSC. To understand the molecular consequences of this amino acid exchange, we developed a model cell line to explore how IleRS biochemistry and functions within the cell would be affected.

Results

Establishment of a c.3521T>A, p.I1174N IleRS model cell line

The amino acid Ile1174 in IleRS is located in the UNE-I domain, which mediates interactions with EPRS within subcomplex III of the MSC (Fig. 1A). We used CRISPR/Cas9-based prime gene editing (40) to introduce the c.3521T>A mutation into a model cell line, human embryonic kidney (HEK) 293T, at the endogenous IARS1 locus (Fig. 1B). To this end, we designed guide RNA (gRNA), repair template, and second nick gRNA and inserted them into pEA1, a plasmid that carries the original Cas9-reverse transcriptase fusion protein (41). HEK 293T cells were then transfected with this plasmid, and GFP-positive cells were sorted into individual wells (Fig. S1A), from which we retrieved 10 colonies (Fig. S1B). The DNA sequence flanking the mutation site was amplified by PCR, and the presence of the edit was verified by Sanger sequencing (Figs. 1C, S1C). We identified five colonies that were heterozygous for the desired edit and four colonies that were homozygous (c.3521T>A, Fig. S1C). Two clones with homozygous edits were selected for follow-up experiments; they will be referred to as I1174N_3 and I1174N_9 in the following.Figure 1**Characterization of a cell line model carrying a disease-causing mutation in IARS1.**A, upper panel, schematic representation of the c.3521T>A point mutation in IARS1 exon 32, which leads to I1174N and the interactions of IleRS with other multisynthetase complex proteins. IleRS forms subcomplex III by binding to LeuRS via its catalytic domain and EPRS through its UNE-I domains. The amino acid I1174 is located in the UNE-I domain. Lower panel, domain organization of IleRS. B, design of prime editing sequences and their location within the IARS1 gene. The mutation involves a codon change from ATC (isoleucine) to AAC (asparagine), highlighted in dark blue within the cyan-shaded codon sequence. IARS1 exon 32 in pink, the repair template, gRNA, and second nick guide RNA in gray, dark green, and light green, respectively. C, Sanger sequencing chromatogram of cells with a c.3521T>A point mutation in IARS1 exon 32, which leads to I1174N. D, RT–quantitative PCR to determine IARS1 mRNA level in human embryonic kidney 293T WT and I1174N cells. IARS1 was normalized to the housekeeping gene GAPDH. Ordinary one-way ANOVA, n = 3, 3, 3. E, representative Western blot probing IleRS protein in HEK 293T wt and I1174N cells and densitometric analysis. Results were normalized against GAPDH. Ordinary one-way ANOVA, n = 3, 3, 3, ∗∗p = 0.028, 0.029. C and D, I1174N_3: IleRS I1174N variant clone 3. gRNA, guide RNA; I1174N_9, IleRS I1174N variant clone 9; IleRS, isoleucyl-tRNA synthetase; UNE, unique.

c.3521T>A mutation affects the IleRS protein but not the mRNA levels

After establishing the cell line, we investigated the impact of the mutation on IARS1 mRNA and protein levels. To assess mRNA levels, we performed a two-step RT–quantitative PCR (qPCR) using primers that span IARS1 exons 26 and 27, positioned upstream of the mutation site. Primer efficiency was validated before the experiment, and GAPDH was used as a normalization control because of its stable expression across conditions. RT–qPCR results indicated no significant difference in IARS1 mRNA levels between wt HEK 293T cells and c.3521T>A homozygous cells (Fig. 1D).

We then measured protein levels by Western blot and found a 70% reduction in IleRS in I1174N homozygous cells compared with wt (Fig. 1E). Thus, while IARS1 mRNA levels remained comparable, the I1174N mutation significantly reduced protein levels. Of note, levels of a shorter IleRS variant remained unchanged (Fig. 1E). As the nucleotides encoding amino acid I1174 are closer to the 3′ UTR of the mRNA, an effect on mRNA translation would be less likely, suggesting that the resulting IleRS protein variant is instead less stable. Incubation with the proteasome inhibitor MG132 increased IleRS I1174N (Fig. S1D) but could not fully restore protein levels to wt.

tRNA aminoacylation, protein synthesis, and cell viability were not reduced in the IleRS I1174N cells

Reduced IleRS levels could lead to a reduction of isoleucyl-tRNA aminoacylation. We used tRNA sequencing (42), which can inform on both tRNA abundance and aminoacylation status: β-elimination of the terminal nucleotide at the tRNA CCA ends can be induced with sodium periodate and borate treatment if the tRNAs are not protected by tRNA aminoacylation (42, 43), which can in turn be quantified by sequencing. We found that there was no decrease in isoleucyl-tRNA aminoacylation in IleRS I1174N cells compared with wt (Fig. 2A). Overall tRNA expression levels were mostly unchanged; however, several isoleucyl-tRNA isoacceptors were reduced in their expression (Fig. S1E, Table S1). Aminoacylation levels of leucyl-tRNA and tyrosyl-tRNA were similarly not reduced in I1174N cells (Fig. S1F). Of note, several tRNAs showed a trend toward higher aminoacylation percentages (Figs. 2A, S1F).Figure 2**tRNA aminoacylation, bulk protein synthesis, and cell viability were not reduced in IleRS I1174N cells.**A, tRNA aminoacylation status relative to wt control, determined by tRNA sequencing. The ratio of read counts of the last nucleotide of the CCA-end and the last nucleotide before the CCA-end is shown relative to wt average for isoleucyl-tRNA isoacceptors. n = 3, 3. B, left panel, Western blot to probe puromycin incorporation, which marks newly synthesized proteins. Right panel, results were normalized against GAPDH. n = 3, 3, 3. C, cell viability and cell proliferation on different days after seeding were assessed with Alamar Blue. n = 4, 4, 4. A–C, wt human embryonic kidney 293T cells. IleRS, isoleucyl-tRNA synthetase; I1174N_3, IleRS I1174N variant clone 3; I1174N_9, IleRS I1174N variant clone 9.

To assess the impact of reduced I1174N levels on bulk protein synthesis, puromycin incorporation assays (44) were used. Puromycin can be integrated into elongating peptide chains, allowing the quantification of newly synthesized proteins by immunoblotting (Fig. 2B). Both wt and I1174N cell lines exhibited comparable signal intensities, suggesting that the I1174N exchange did not significantly affect the synthesis of new proteins. We further measured cell viability with Alamar Blue and found that proliferation rates were similar between wt and IleRS I1174N cells, with no significant difference over 72 h (Fig. 2C). The reduction of IleRS protein levels, and thus catalytic capacity per se, was therefore not limiting for cell viability.

I1174N disrupts MSC formation

Since the UNE-I domain is crucial for the integration of IleRS into the MSC (39, 45), we hypothesized that the I1174N substitution might disrupt the IleRS association with other components in the complex. To investigate the overall integrity of the MSC as well as the presence of IleRS, we performed size-exclusion chromatography (SEC) followed by Western blot analysis. Under mild cell lysis conditions, the MSC stays intact (46), and proteins in fractions corresponding to different molecular weights can be detected through immunoblotting. We probed MSC-bound aaRSs (IleRS, ArgRS, and LeuRS) and non-MSC–bound TyrRS to assess whether IleRS was still bound in the MSC and would therefore elute at earlier fractions. Immunoblotting of wt cell lysates separated into SEC fractions (Fig. 3A, quantified in Fig. S2A) showed a prominent IleRS signal at fractions 9 to 10, in line with MSC association. IleRS levels in I1174N cells were overall lower, leading to lower signal in fractions 9 to 10 (Fig. 3A), but the remaining full-length IleRS was still found in MSC-associated fractions (Fig. S2A). Due to the reduction of full-length IleRS, lower molecular weight isoforms of IleRS, eluting at fraction 11 (which were also present in wt), constitute a larger percentage of total IleRS protein in I1174N (Figs. 3A, S2A). These variants could be splice isoforms or proteolytic fragments, but we were not able to unambiguously assign them.Figure 3**Isoleucyl-tRNA synthetase (IleRS) I1174N caused the exclusion of IleRS and leucyl-tRNA synthetase (LeuRS) from the multisynthetase complex.**A, cell lysate separated by size-exclusion chromatography to distinguish between monomeric/dimeric and multisynthetase complex–bound aminoacyl-tRNA synthetases (aaRSs) followed by Western blot. Intact multisynthetase complex elutes between fractions 9 and 11. Dimeric or monomeric tRNA synthetases elute between fractions 13 and 15. Western blots are shown as a representative of at least three biological replicates. B, Volcano plot of proteins identified by mass spectrometry following coimmunoprecipitation. Y-axis: significance (p value). X-axis: difference (fold change). Color coding denotes the number of peptides per protein. Upper panel, IleRS wt interactome (anti-IleRS antibody pulldown compared with IgG control pulldown in wt cell line). Middle panel, IleRS I1174N interactome (anti-IleRS antibody pulldown compared with IgG control pulldown in I1174N cells). Lower panel, comparison between wt and IleRS I1174N interactomes (anti-IleRS antibody pulldowns in wt and I1174N lysates). Protein enriched in wt IleRS pulldowns but not in IleRS I1174N appears on the left side of the volcano plot. n = 3, 3. A and B, wt human embryonic kidney 293T cells. ArgRS, arginyl-tRNA synthetase; I1174N_9, IleRS I1174N variant clone 9; TyrRS, tyrosyl-tRNA synthetase.

We next explored the influence of IleRS I1174N on proteins in other MSC subcomplexes. ArgRS is still associated with the MSC, suggesting that the majority of the MSC is still intact (Figs. 3A, S2A). However, full-length ArgRS bands increased in fractions corresponding to free-standing ArgRS, indicating that the MSC might be less stable than in wt cells (Figs. 3A, S2A).

LeuRS is tethered to the MSC through IleRS, and we hypothesized that LeuRS would dissociate if the relative amount of IleRS in the MSC is reduced. In wt cells, full-length LeuRS was mostly detected in fractions 9 to 10, whereas in I1174N cells, LeuRS additionally appeared in comparable intensity in fractions 13 and 14 (Figs. 3A, S2A). These fractions correspond to free-standing LeuRS, suggesting a significant decrease of MSC-bound LeuRS in I1174N cells. This indicates that the reduced integration of IleRS into the MSC in turn affected LeuRS. TyrRS eluted consistently between fractions 13 and 15 in both cell lines, as expected for a non-MSC–bound protein (Fig. 3A).

We further mapped IleRS protein–protein interactions through interactome studies. To this end, we coimmunoprecipitated IleRS from both wt and I1174N cells and identified interaction partners through mass spectrometry (MS; Fig. 3B, Tables S2 and S3). Pulldown of IleRS and enrichment of its known interaction partner LeuRS was verified with Western blot (Fig. S2B). We retrieved an unexpectedly large number of interactors and opted to repeat the coimmunoprecipitation with a second polyclonal antibody to control for possible antibody-off target effects (Fig. S2, C and D, Table S3). While in both experiments, additional interactors were found, only members of the MSC were present in both (Fig. S2E).

In wt cells, the IleRS interactome included all MSC components, except GlnRS, for which insufficient peptides were detected (Fig. 3B, upper panel). GlnRS was present in pulldowns with the second IleRS antibody (Fig. S2D). In contrast, in I1174N cells, IleRS enrichment only retrieved LeuRS, whereas all other MSC components were absent (Fig. 3B, middle panel). Comparison between both interactomes suggested that the main interactors lost in IleRS I1174N were MSC-bound aaRS (Fig. 3B, lower panel).

Using the alternative antibody, IleRS enrichment was reduced in I1174N cells, and the LeuRS interaction was also reduced along with other MSC components (Fig. S2D), which could either be due to a difference in antibody-binding site or reflective of lower IleRS enrichment in this experiment. This confirms our SEC results, suggesting again that the I1174N exchange lowered the overall amount of IleRS available for association with MSC proteins. As the levels of shorter IleRS isoforms remained mostly unchanged or increased compared with full-length IleRS, they now constitute a higher percentage of IleRS, leading to an overall reduction of full-length, MSC-bound IleRS, which is reflected in the IleRS interactome.

IleRS I1174N affects levels of the stress response protein activating transcription factor 4

In order to understand how the I1174N variant might affect cellular processes, we tested several cell signaling pathways that were previously associated with aaRS variants and disease progression: mutations in aaRSs are known to cause changes in the integrated stress response, most commonly its increased activation (47, 48, 49). As the integrated stress response reports on uncharged tRNAs and perturbations in amino acid homeostasis (50), it is specifically vulnerable to alterations in tRNA aminoacylation. We therefore probed for activating transcription factor 4 (ATF4) protein levels, as ATF4 is a central regulator of the integrated stress response (51, 52). Immunoblotting was conducted on cells subjected to different cell stress conditions for 24 h to exacerbate ATF4 modulation: low glucose, hypoxia mimicry, and serum starvation (53) (Fig. 4A). At baseline, in untreated cells, ATF4 levels were higher in wt than in I1174N cells (Fig. 4A). This reduction persisted in low-glucose medium, where ATF4 levels in I1174N cells were lower than those in wt cells (Fig. 4A). Notably, levels of phosphorylated eukaryotic initiation factor 2A were unchanged (Fig. S3A). Total protein synthesis in reduced glucose medium, visualized by puromycin incorporation, was unchanged, suggesting that the altered ATF4 level did not impact bulk protein synthesis (Fig. 4B). In other stress conditions, such as hypoxia mimicry and serum starvation, ATF4 levels were comparable to wt or showed a higher trend (Fig. 4A).Figure 4**Reduced induction of integrated stress response regulator ATF4 in low-glucose medium in IARS1 I1174N cells.**A, upper panel, protein levels of ATF4 were analyzed by Western blot following 24 h of varying cell stress conditions. Hypoxia, hypoxia mimicry through the addition of the iron chelator DFO. Low glucose, low-glucose medium. w/o serum, serum starvation through removal of FBS. Hsp90 was used as a loading control. Lower panel: densitometric quantification, n = 3, 3. B, Western blot to probe puromycin incorporation in low-glucose medium, which marks newly synthesized proteins. C, upper panel, Western blot probing ATF4 levels over time in low-glucose medium. Lower panel, densitometric analysis. n = 3, 3. A–C, wt human embryonic kidney 293T cells. A representative of at least three biological replicates is shown for Western blots. ATF4, activating transcription factor; DFO, deferoxamine; I1174N_9, IleRS I1174N variant clone 9.

To investigate how fast a difference in ATF4 regulation would be detectable in low-glucose medium, we tested ATF4 levels at different time points after exposing cells to reduced glucose levels. At basal, high glucose levels, ATF4 protein was already lowered in I1174N cells (Fig. 4C). At 4 h, 8 h, and 16 h after glucose starvation, a reduction in ATF4 levels was observed in cells with the I1174N IleRS variant (Fig. 4C). To test whether ATF4 target gene expression was changed, we used qPCR to assess levels of CHOP, HSPA5, EIF2S2, EPRS, YARS1 (TyrRS), and SARS1 mRNA after 24 h in low-glucose medium. While we would expect downregulation of ATF4 target genes, we did not find consistent changes (Fig. S3B). We also explored stress granule formation, as indicated by condensates marked with G3BP1, which are connected to the integrated stress response in many ways (54, 55). Low-glucose medium was insufficient to cause stress granule formation after 24 h (Fig. S4, A and B). To exacerbate stress conditions, we exposed cells to arsenite for 1 h, which led to the formation of G3BP1 condensates (Fig. S4, A and B). Similar numbers of G3BP1 punctae and G3BP1-positive areas were found for both wt and I1174N cells. Cell proliferation did not change in response to low glucose (Fig. S4C).

The mTOR pathway controls the cellular response to nutrients, such as amino acids. As LeuRS was reduced in the MSC (Fig. 3A) and LeuRS protein levels were reduced in I1174N cells (Fig. S4D), we explored the LeuRS-controlled mTOR signaling pathway, which integrates amino acid metabolism with the response to glucose fluctuation (31, 56). We therefore quantified phosphorylation levels of the mTOR downstream target ribosomal protein S6 kinase 1 (S6K1) (Fig. S4E). Levels of phosphorylated S6K1 dropped over time in wt cells, and low-glucose medium exacerbated this effect (Fig. S4E). I1174N cells, however, showed more steady phosphorylated S6K1 levels after 8 h in comparison to wt because of a slower decrease over time (Fig. S4E), suggesting that the cellular response to low glucose is attenuated in I1174N cells.

Discussion

Here, we evaluate the impact of the disease-causing IleRS I1174N variant on its integration into the MSC, its effects on protein synthesis and cell growth, the IleRS interactome, and cellular stress signaling. We found that the I1174N amino acid exchange strongly decreased IleRS protein levels, whereas mRNA levels remained constant. The resulting reduction of IleRS did not negatively affect tRNA aminoacylation levels, bulk protein synthesis, or cell viability. However, fewer IleRSs were incorporated into the MSC, and in turn, increased the amount of free LeuRS. Levels of the integrated stress response regulator ATF4 were reduced at baseline and in low-glucose medium.

In the affected individual (35), I1174N is accompanied by another mutation on the other allele, c.1252C>T,p.418R>∗, which truncates IleRS within its catalytic domain and deletes the IleRS tRNA-binding domain, very likely resulting in a catalytically inactive protein. Neither variant by itself was disease causing, as evidenced by the unaffected parents and siblings (35), suggesting that the activity resulting from one allele is sufficient to support aminoacylation needs. The effects of I1174N and R418∗ therefore seem additive, together lowering IleRS functionality to a critical degree. Homozygous cases with I1174N have not been reported, and it is difficult to predict whether IleRS I1174N alone would be sufficient to elicit a disease phenotype. We opted to explore a homozygous cell line model to allow focused and unambiguous studies on the biochemical and cell biology consequences of the I1174N variant.

The mammalian MSC can be divided into three subcomplexes (14): subcomplex III is a trimeric complex composed of EPRS, IleRS, and LeuRS (Fig. 1A). Within this structure, IleRS interacts with EPRS via its UNE-I domain and anchors LeuRS, which binds to the catalytic domain of IleRS through its UNE-L domain. EPRS then facilitates the connection between subcomplexes II and III. In the cocrystal structure of the chicken EPRS–IleRS complex (Protein Data Bank code: 7WRS) (33), I1174 is not directly in contact with EPRS, and the closest contact to EPRS is over 10 Å away. AlphaFold places amino acid 1174 in a beta sheet in the middle of the human UNE-I domain, and the Ile-to-Asn exchange is not predicted to disrupt its fold. As EPRS binds in a cleft between the two UNE-I domains (33), the I1174N exchange might however disrupt the careful spacing between the two or otherwise indirectly interfere with protein–protein interactions.

The integration of IleRS into the MSC can not only influence IleRS functionality but also protein stability: A previous study has shown that the levels of aaRSs in the MSC can be dependent on each other, as knockdown of individual components led to a depletion of others (45). Reduction of the adapter proteins, AIMP1 and AIMP2, as well as EPRS, led to decreased IleRS levels (45), suggesting that IleRS outside the MSC could be less stable. In turn, IleRS knockdown also showed lower levels of MetRS and LeuRS (45), which we could confirm for LeuRS (Fig. S4D). The reduction of IleRS observed here could result from lowered stability of the mutant protein itself or as a secondary effect following dissociation from the MSC, potentially leading to the prompt degradation of free-standing full-length IleRS. The remaining full-length IleRS was predominantly bound to the MSC, but shorter IleRS isoforms constituted a larger percentage of total IleRS. Future studies will aim to explore how IleRS protein levels are regulated, as mRNA stability seems unchanged.

Previous studies on the role of the MSC suggest that its functions might be multifaceted: a channeling effect of the MSC, meaning the increased flow of charged tRNAs to the ribosome, has been discussed (57). In addition, specific genes are differentially translated upon exclusion of ArgRS and GlnRS from the MSC (21), and the MSC could affect protein synthesis differently across transcripts. However, bulk translation appears to be less sensitive to the disruption of MSC formation (58). We found that bulk protein synthesis was similarly unaffected by the reduction of IleRS levels and its altered MSC association (Fig. 2B). In line with this, cell viability was unaltered in I1174N cells, and proliferation rates were comparable to wt cells (Fig. 2C). Cell stress through low glucose did not exacerbate this phenotype as protein synthesis was likely still sufficient.

The integrated stress response mediates the cellular adaptation to different environmental triggers, such as dsRNA exposure, signifying viral infection, accumulation of unfolded proteins, and amino acid starvation (50). New studies suggest that the integrated stress response can take different forms (54), which may explain the absence of increased eukaryotic initiation factor 2α phosphorylation concomitant with elevated ATF4 levels here. Overactivation of the integrated stress response is a strong contributor to aaRS mutation–induced Charcot–Marie–Tooth disease, a neurodegenerative disorder, which predominantly affects long peripheral motor neurons (59). In GARS1 Charcot–Marie–Tooth mutations, sequestration of tRNAs by disease-causing GlyRS variants induced an overactivation of the integrated stress response (47, 48, 49). Here, we found the opposite, where a disease-causing variant of IleRS led to reduced baseline levels and activation of ATF4, which persisted in low-glucose medium (Fig. 4, A and C). Of note, other stressors led to a trend toward increased ATF4 levels, in line with other reports on disease-causing aaRS mutations (Fig. 4A) (47, 48, 49).

The integrated stress response can be triggered by uncharged tRNAs and ribosome collision (60, 61), and we did not observe less tRNA charging in I1174N cells, despite lowered IleRS protein levels (Fig. 2A). A recent study showed that inhibition of aaRS enzymatic activity prevented ribonucleoprotein granule assembly upon exogenous stress (62). We did not find that G3BP1-marked stress granules were changed, even under severe oxidative stress, suggesting again that IleRS activity was not sufficiently lowered in I1174N cells to abrogate stress granule formation. Alterations in ATF4 levels in complete cell culture medium and under mild nutrient deprivation could indicate defects in the cellular adaptation to stress, which may be more severe in tissue cell types that rely predominantly on glucose as an energy source, such as cells in the brain (63). This could tie our observed changes to phenotypic aberrations leading to developmental disorders.

In summary, we report the marked reduction of IleRS protein in a cell line modeling a disease-causing variant together with the exclusion of IleRS from its native protein complex. These observations went hand in hand with an impaired induction of the integrated stress response in a nutrient-low environment. This suggests that reduced cellular adaptation to stress could be a potential factor in IleRS-driven disease elicited by mutations in IARS1.

Experimental procedures

Cell culture

HEK 293T cells were obtained from the American Type Culture Collection and confirmed by short tandem repeat analysis. Cells were cultured at a density of 80% to 90% confluency and passaged on average twice per week. If not stated otherwise, cells were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Sigma–Aldrich), with 10% fetal bovine serum (FBS) (Corning) and 1% penicillin–streptomycin (Sigma–Aldrich). Cells were tested monthly for mycoplasma contamination and were consistently found to be negative. Experiments were performed before passage 30.

Prime editing and single-cell sorting

The PEA1-GFP plasmids were a gift from Paul Thomas (Addgene #171993) (41). gRNA, repair template, and second nick gRNA were designed using Petal (41). Sequences are shown in Figure 1B. The plasmid was transfected into HEK 293T cells using Calfectin (SignaGen). Successfully transfected cells were selected based on GFP expression by the Flow Cytometry Facility of the Temerty Faculty of Medicine and sorted as individual cells into a 96-well plate.

Sanger sequencing

DNA was isolated with the DNA extract solution containing proteinase K (QuickExtract DNA Extraction Solution; Lucigen). Primers were designed to amplify 596 bp surrounding the c.3521T>A site in a two-step PCR using Phusion polymerase (New England Biolabs [NEB]). The molecular weight of the amplified product was confirmed by 1% agarose gel electrophoresis. The product was then purified with a PCR purification kit (FroggaBio), and 300 ng of DNA were sequenced with a custom primer at The Centre for Applied Genomics, SickKids, Toronto.

Protein extraction

Cells were seeded at a density of 5 × 10^6^ cells into 10 cm dishes 1 day prior to harvest. After washing with PBS (Sigma–Aldrich) twice, total protein was collected in 500 μl radioimmunoprecipitation assay lysis buffer (0.05 M Tris–HCl, pH = 7.4, 0.15 M NaCl, 0.25% deoxycholate, 1% NP-40, and 1 mM EDTA) with protease inhibitor cocktail (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail; Roche). Cells underwent freeze–thaw cycles to ensure complete lysis and were stored at −80 °C. Lysates were centrifuged at 14,000g for 10 min, and the pellet was removed. SDS-loading buffer (5x: 0.25% bromophenol blue, 0.5 M DTT, 50% glycerol, 10% SDS, and 0.25 M Tris–HCl) was added, and the lysates were heated at 95 °C for 7 min prior to SDS-PAGE.

Western blot

Total cell lysates were quantified using a bicinchoninic acid assay, and 3 to 5 μl (6–10 μg protein) of lysates were loaded on 4% to 12% gradient gel for gel electrophoresis. Transfer to polyvinylidene fluoride membranes was performed with the Invitrogen iBlot 2 Dry Blotting system. Membranes were blocked in 3% milk/Tris-buffered saline with Tween-20 (TBST) for 1 h. After washing with TBST, membranes were incubated with the respective primary antibody in 5% bovine serum albumin/TBST overnight at 4°. The next day, membranes were washed three times for at least 10 min with TBST. Membranes were then incubated with the corresponding horseradish peroxidase–conjugated secondary antibody diluted in 1% milk/TBST (Goat-anti-rabbit, Invitrogen, 1:5000 dilution; Goat-anti-mouse, Invitrogen, 1:10,000 dilution) for 60 to 90 min and washed. Western blots were visualized with chemiluminescence (SignalFire ECL Reagent; Cell Signaling) and imaged with a ChemiDoc (Bio-Rad). All antibodies used here are listed in Table S4 and were verified by the manufacturer. Loading was assessed using housekeeping proteins as indicated in the respective figures. Western blots were quantified either using ImageJ or Image Lab Software (Bio-Rad).

RNA isolation

Cells were seeded into 10 cm dishes 2 days prior to the harvest at a density of 3 × 10^6^ cells/dish. After washing with PBS (Sigma–Aldrich) twice, RNA was extracted with TRIzol (Invitrogen) following the manufacturer's instructions. RNA integrity was confirmed by 1% agarose gel electrophoresis.

RT–qPCR

Complementary DNA (cDNA) was synthesized from extracted RNA using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen) according to the manufacturer's instructions. A LightCycler 480 System (Roche) was used to analyze cDNA levels, and the values were normalized to GAPDH/18S. 2^-ΔΔCt^ values are shown. All the qPCR primers used here can be found in Table S5.

SEC to assess protein complex formation and integrity

Cells were seeded into 15 cm dishes 1 day prior to the experiment at a density of 5 × 10^6^ cells/dish. After washing with PBS (Gibco) twice, cells were harvested by adding 500 μl MSC lysis buffer (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% NP-40, and cOmplete Protease Inhibitor Cocktail Mini, EDTA-free Protease Inhibitor Cocktail, Roche). The lysates were incubated on ice for 20 min, followed by a 10-min spin at 14,000g to obtain soluble proteins. The cell lysates (500 μl) were then injected onto a Superdex 200 Increase Tricorn 10/300 GL Prepacked SEC Column (Cytiva) equilibrated with PBS (Gibco), and 1 ml fractions were collected. Each fraction (20 μl) was analyzed by Western blot.

tRNA library preparation and sequencing analysis

Multiplex small RNA sequencing libraries were prepared as described by Watkins et al. (42), with minor modifications. Total RNA was extracted from wt HEK 293T and I1174N-9 HEK 293T cells using TRIzol (Invitrogen), and 1 μg of total RNA per sample was used as input. RNA was kept under acidic conditions to preserve the aminoacylation status.

To preserve tRNA aminoacylation information, only the acylated multiplex small RNA sequencing workflow was performed. Total RNA was subjected to one-pot periodate oxidation and β-elimination to selectively remove the terminal 3′ nucleotide from uncharged tRNAs, followed by 3′ end repair with T4 polynucleotide kinase (NEB). Barcoded biotinylated capture hairpin oligonucleotides were ligated to RNA 3′ ends using T4 RNA ligase I (NEB), after which samples were pooled and immobilized on streptavidin-coated MyOne C1 Dynabeads (ThermoFisher).

All subsequent steps were performed on-bead, including dephosphorylation, reverse transcription using SuperScript IV VILO (ThermoFisher) under extended incubation conditions, RNase H (NEB) digestion, periodate oxidation to inactivate unligated adapters, and second adapter ligation. Libraries were PCR amplified using Q5 high-fidelity DNA polymerase, size selected by 10% Tris–borate–EDTA PAGE (Invitrogen) to remove terminal transferase byproducts, and purified by gel extraction followed by ethanol precipitation.

Final libraries were sequenced on a NovaSeq X using paired-end sequencing. Sequencing data were analyzed as described by Frietze and Pan (64), with support from Trillium high-performance computing, operated by the University of Toronto and Compute Canada. In brief, paired-end reads were merged, aligned to human tRNA sequences (64) with Bowtie2 (65), and reads at each nucleotide position were quantified using the pileup function of Samtools (66). Read counts per tRNA were quantified with Pysam as outlined (64), and differential expression was assessed with DESeq2 (67).

tRNA sequencing data are deposited at Gene Expression Omnibus with GEO ID GSE315548.

Puromycin incorporation assay

Assays were performed as described (44). In brief, cells were seeded into a 6-well plate 1 day prior at a density of 5 × 10^5^ cells/well. Puromycin was directly added at 10 μg/ml for 30 min (pulse). The medium was then changed (chase), and cells were allowed to recover for an additional hour before lysis in radioimmunoprecipitation assay buffer and analysis by Western blot.

Cell proliferation assay

Cells were seeded in quadruplets in 96-well cell culture plates at 5000 cells/well. One plate was used for every time point. Alamar blue reagent (10%; Invitrogen) was added 2 h prior to fluorescence intensity measurements (λ_ex_ = 570 nm, λ_em_ = 600 nm) with a microplate reader (Synergy H1; Biotek).

Coimmunoprecipitation

293T wt or I1174N mutant cells were seeded onto 15 cm dishes at a density of 15 × 10^6^ cells per dish, 1 day prior to lysis. Cells were harvested in 1 ml of mild lysis buffer (Tris-buffered saline with 1% NP-40 and 1 mM MgCl_2_). Cells were incubated on ice for 30 min, followed by centrifugation at 14,000g for 20 min to remove insoluble components. The supernatant (1 ml) was loaded onto 30 μl of pre-equilibrated protein A/G agarose beads (SCBT) along with 3 μl anti-IleRS antibody (Proteintech or Bethyl Laboratories). After 3 h, beads were washed once with 1 ml 0.05% NP-40 in TBS and twice with 1 ml TBS. The beads were then stored in PBS (Gibco) at −80 °C.

Protein digestion and MS

MS experiments were performed as described (68). Peptides were eluted from protein A/G agarose beads by trypsin digest at room temperature overnight (∼16 h) while shaking (5 ng/μl trypsin, 2 M urea, 50 mM Tris–HCl, pH 7.5, 0.2 mM DTT, and 1 mM chloroacetamide). The digested peptides were desalted with C18 pipette tips (Pierce) according to the manufacturer's instructions. Peptides were eluted in a final volume of 30 μl, concentrated using a SpeedVac (Thermo Fisher), and stored at −80 °C. Prior to analysis, peptides were resuspended in 40 μl of 1% formic acid (FA).

Samples were analyzed using an EASY-nLC 1200 UHPLC system combined with a Q-Exactive HF-X Orbitrap mass spectrometer via an in-line nanoLC-electrospray ion source (Thermo Fisher Scientific). Solubilized peptides were loaded onto a house-made fused-capillary silica precolumn (100 μm I.D.) packed with 2 cm of 5 μm Luna C18 100 Å reverse-phase particles (Phenomenex) and separated on a 14.1 cm (100 μm I.D.) silica pulled emitter packed with 1.9 μm Luna C18 100 Å reverse-phase particles (Phenomenex); the capillary was sourced from Polymicro Technologies. Peptides were eluted over 120 min with mobile phase A (0.1% FA) and mobile phase B (80/20/0.1 acetonitrile/water/FA) at a constant flow rate of 300 nl/min with a linear increase from 2% to 5% B over 1 min, an increase to 26% B over 70 min, an increase to 60% B over 20 min, then an increase to 100% B over 14 min, and a final plateau at 100% B for 15 min.

MS was performed, as described by Hall et al. (69). In brief, a data-dependent top 20 method was used. Full MS scans were acquired from m/z 350 to 1400 at a resolution of 60,000 at 200 m/z with a target automated gain control of 3 × 10^6^ charges. For higher-energy collisional-dissociation MS/MS scans, the normalized collision energy was set to 28 and a resolution of 15,000 at 200 m/z. Precursor ions were isolated in a 1.4 m/z window and accumulated for a maximum of 20 ms or until the automatic gain control target of 1 × 10^5^ ions was reached. Precursors with unassigned charge states, a charge of 1+, or a charge of 7+ and higher were excluded from sequencing. Previously targeted precursors were dynamically excluded from resequencing for 20 s.

MS data were processed with MaxQuant 2.6.4.0 and Perseus 2.1.2 (70, 71, both were developed at the Max Planck Institute of Biochemistry), as described previously (68), to identify IleRS interaction partners. In brief, raw files were searched against the Homo sapiens reference proteome UP000005640_9606 (72) (UniProt) with the Andromeda search engine integrated into MaxQuant: M oxidation, N-terminal acetylation as variable; cysteine carbamidomethyl as fixed modification; and two missed cleavages maximum. First search peptide tolerance = 20 ppm; main search peptide tolerance = 4.5 ppm; protein false discovery rate = 0.1; and minimum peptides = 1. Default settings were used for label-free quantification (minimum ratio count = 2). The resulting protein groups file was loaded into Perseus and filtered for “reverse”, “potential contaminants,” and “only identified by site.” The log2 values of label-free quantification intensities were calculated, samples were grouped according to experimental conditions (wt, IARS1 mutant), and all proteins with less than two valid values/group were discarded. Missing values were replaced from a normal distribution (width 0.3 and downshift 1.8), and a two-tailed unpaired Student's t test was used to calculate t test significance and difference (fold change) for each protein (71) by comparing wt versus IleRS1 I1174N interactomes. Data can be accessed through PRIDE with the project accession PXD067475.

Induction of cell stress and proteasome inhibition

Cells were seeded into a 6-well plate at a density of 1 × 10^6^ cells per well, 1 day prior to treatment. Cells were washed with PBS (Gibco) before introducing fresh medium. The control group was maintained in high-glucose DMEM (Sigma–Aldrich) supplemented with 10% FBS (Corning) and 1% penicillin–streptomycin (Sigma–Aldrich). To stabilize IleRS, cells were incubated with 10 μM MG132 in dimethyl sulfoxide or a dimethyl sulfoxide control, respectively. To mimic the hypoxia group, 100 μM deferoxamine (Sigma–Aldrich) was added. Cells in serum-free conditions were exposed to DMEM without FBS and low-glucose DMEM (Sigma–Aldrich) with 10% FBS, and 1% penicillin–streptomycin was used for low-glucose conditions. Cells were exposed to stressors for the indicated time and then harvested for Western blot analysis.

Immunofluorescence

293T wt and I1174N mutant cells were cultured on poly-d-lysine (Sigma–Aldrich, P7280-5MG)-coated coverslips (Electron Microscopy Science, #72230-01, 12 mm). For stress treatments, cells were subjected to either a low-glucose condition by culturing in low-glucose DMEM for 24 h at 37 °C or treated with 0.5 mM sodium arsenite (Sigma–Aldrich, S7400) for 1 h at 37 °C.

Following that, cells were washed with PBS and fixed in 4% paraformaldehyde (BioShop, PAR070) for 15 min at room temperature. After fixation, cells were permeabilized with 0.25% Triton X-100/PBS for 5 min at room temperature, then blocked with 1% bovine serum albumin (BioShop, 9048-46-8) and 2% goat serum (Corning) in PBS for 1 h at room temperature. Primary antibodies against G3BP1 were diluted in blocking solution and incubated with the cells overnight at 4 °C. The next day, cells were washed in blocking buffer three times for 5 min and incubated with DyLight 650 secondary antibody (1:200 dilution) for 2 h at room temperature, protected from light. To counterstain DNA, cells were incubated with 4′,6-diamidino-2-phenylindole (Roche, 10236276001) for 15 min at room temperature, followed by three PBS washes. Coverslips were briefly rinsed with distilled water and mounted on glass slides using ProLong Gold Antifade Reagent (Cell Signaling Technologies, #9071S) overnight at room temperature, protected from light. Samples were sealed with nail polish and stored at 4 °C in the dark chamber before imaging. All antibodies are listed in Table S3.

Confocal microscopy

Images were captured using a Leica TCS SP8 confocal microscope (Leica), with 63×/1.40 Oil HC PL APO CS2 objective and Leica Microsystem's LAS X software. Coverslips were mounted with Type F Immersion Liquid (Leica). Images were taken at 1024 × 1024 with identical acquisition parameters across samples, processed with ImageJ/Fiji, and exported as TIFF files. Quantification of stress granules was performed using ImageJ/Fiji.

Statistics

Biological replicates are different cell passages. Statistical significance was evaluated by comparing biological replicates with either an unpaired Student's t test or ANOVA, respectively (GraphPad Prism). Bar graphs show the mean with the standard deviation. MS data were analyzed using Perseus (71).

Data availability

tRNA sequencing data are deposited at Gene Expression Omnibus and can be accessed with GEO ID GSE315548. IleRS interactomes can be accessed through PRIDE with project accession number PXD067475 reviewer_pxd067475@ebi.ac.uk∖. Cell lines and plasmids are available upon request to haissi.cui@utoronto.ca.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.