A Translation-Independent Function of Phers Activates Growth and Proliferation in Drosophila Manh Tin Ho*,§, Jiongming Lu‡,§, Dominique Brunßen and Beat Suter¶

RESEARCH ARTICLE A translation-independent function of PheRS activates growth and proliferation in Drosophila Manh Tin Ho*,§, Jiongming Lu‡,§, Dominique Brunßen and Beat Suter¶

ABSTRACT not been studied. This might have been due to the assumption that Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) not only load higher PheRS levels could simply reflect the demand of tumorigenic the appropriate amino acid onto their cognate tRNAs, but many of cells for higher levels of translation, or it could have to do with the them also perform additional functions that are not necessarily related difficulty of studying the moonlighting function of a protein that is to their canonical activities. Phenylalanyl tRNA synthetase (PheRS/ essential in every cell for basic cellular functions such as translation. FARS) levels are elevated in multiple cancers compared to their Aminoacyl tRNA synthetases (aaRSs) are important enzymes normal cell counterparts. Our results show that downregulation of that act by charging tRNAs with their cognate amino acid, a key PheRS, or only its α-PheRS subunit, reduces organ size, whereas process for protein translation. This activity makes them essential elevated expression of the α-PheRS subunit stimulates cell growth for accurately translating the genetic information into a polypeptide and proliferation. In the wing disc system, this can lead to a 67% chain (Schimmel and Söll, 1979). Besides their well-known role in increase in cells that stain for a mitotic marker. Clonal analysis of twin translation, an increasing number of aaRSs have been found to spots in the follicle cells of the ovary revealed that elevated perform additional functions in the cytoplasm, the nucleus and even expression of the α-PheRS subunit causes cells to grow and outside the cell (Guo and Schimmel, 2013; Nathanson and proliferate ∼25% faster than their normal twin cells. This faster Deutscher, 2000; Smirnova et al., 2012; Casas-Tintó et al., 2015; growth and proliferation did not affect the size distribution of the Gomard-Mennesson et al., 2007; Greenberg et al., 2008; Otani proliferating cells. Importantly, this stimulation proliferation turned out et al., 2002; Zhou et al., 2014). Moonlighting aaRSs regulate to be independent of the β-PheRS subunit and the aminoacylation alternative splicing, RNA processing and angiogenesis (Lee et al., activity, and it did not visibly stimulate translation. 2004). For example, the amino acid-binding site of LysRS has an immune response activity, and TrpRS inhibits vascular endothelial This article has an associated First Person interview with the joint first (VE)-cadherin and through this elicits anti-angiogenesis activity authors of the paper. (Tzima et al., 2005; Yannay-Cohen et al., 2009). Cytoplasmic PheRS is one of the most complex members of the KEY WORDS: Aminoacyl tRNA synthetase, PheRS, FARSA, aaRSs family, a heterotetrameric protein consisting of two alpha (α) Growth and proliferation control and two beta (β) subunits responsible for charging tRNAPhe during translation (Roy and Ibba, 2006). The α-subunit includes the INTRODUCTION catalytic core of the tRNA synthetase, and the β-subunit has Many cancer tissues display higher levels of phenylalanyl transfer structural modules with a wide range of functions, including tRNA RNA (tRNA) synthetase (PheRS; also known as FARS) than their anti-codon binding, hydrolyzing mis-activated amino acids and healthy counterparts according to the database ‘Gene Expression editing misaminoacylated tRNAPhe species (Ling et al., 2007; Lu across Normal and Tumor tissues 2’ (GENT2) (Park et al., 2019). et al., 2014; Roy and Ibba, 2006). Importantly, both subunits are Interestingly, a correlation between tumorigenic events and PheRS needed for aminoacylation of tRNAPhe. expression levels had been noted much earlier for the development We set out to address the question whether and how elevated levels of myeloid leukemia (Sen et al., 1997). Despite this, a possible of PheRS can contribute to tumor formation. To test for this activity, causative connection between elevated PheRS levels and tumor we studied the role of PheRS levels in the Drosophila model system, formation had so far not been reported and, to our knowledge, also with the goal of finding out whether elevated levels of PheRS allow higher translation activity or whether a moonlighting role of PheRS might provide an activity that contributes to elevated growth and Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Bern 3012, proliferation. We found that α-PheRS levels regulate cell proliferation Switzerland. *Present address: Department for BioMedical Research (DBMR), University of in different tissues and cell types. Interestingly, however, elevated Bern, Murtenstrasse 35, 3008 Bern, Switzerland. levels of α-PheRS do not simply allow higher levels of translation. ‡ Present address: Max Planck Institute for Biology of Ageing, Joseph-Stelzmann- Instead, α-PheRS performs a moonlighting function by promoting Str. 9b, 50931 Köln, Germany. §These authors contributed equally to this work proliferation independent of the β-PheRS subunit, even if it lacks the aminoacylation activity. ¶Author for correspondence ([email protected])

M.T.H., 0000-0003-1608-2234; J.L., 0000-0002-8879-4938; B.S., 0000-0002- RESULTS 0510-746X PheRS is needed for proliferation and for normal organ

This is an Open Access article distributed under the terms of the Creative Commons Attribution and animal growth License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, The Drosophila FARS homolog PheRS is a hetero-tetrameric aaRS distribution and reproduction in any medium provided that the original work is properly attributed. consisting of two α- and two β-subunits encoded by α-PheRS and β Handling Editor: Ross Cagan -PheRS, which are essential genes in Drosophila (Lu et al., 2014).

Received 29 October 2020; Accepted 21 January 2021 To find out whether cellular levels of α-PheRS correlate with and Disease Models & Mechanisms

1 RESEARCH ARTICLE Disease Models & Mechanisms (2021) 14, dmm048132. doi:10.1242/dmm.048132 possibly contribute to growth, we tested whether reduced levels in presumably because of its role in systemic growth (Texada et al., specific tissues affect growth of the organ and animal. For this we 2020), the fat body knockdown of PheRS reduced the size of the used RNA interference (RNAi) to reduce their activity in two entire pupae (Fig. 1B). specific tissues: the eye, an organ that is not essential for viability, To further analyze the changes at the cellular level, the effect of and the fat body (Fig. 1A,B). Indeed, knocking down either of the knocking down α-PheRS and β-PheRS in Drosophila Kc cells was two subunits in the developing eye reduced the size of the adult eye first examined at the level of cell proliferation (Fig. 1C). The (Fig. 1A). Similarly, reducing α-PheRS or β-PheRS expression knockdowns were carried out by adding double-stranded RNA levels in the larval fat body caused a growth reduction. However, (dsRNA) into the medium, and the cell numbers were recorded over

Fig. 1. PheRS knockdown reduces cell proliferation and tissue size. (A,B) RNAi knockdown of PheRS subunits in fly eyes (A) and fat bodies (B). ey-Gal4 (ey- Gal4/+; UAS-PheRSRNAi/+; A) and ppl-Gal4 (ppl-Gal4/+; UAS-PheRSRNAi/+; B) were used to drive RNAi expression. The controls were GFP RNAi. RNAi knockdown of either subunit reduced the eye size (A; scale bar: 250 µm) and the size of the entire pupae (B; scale bar: 500 µm). (C-E) Proliferation, cell cycle distribution and cell size analysis of Kc cells upon knockdown of PheRS. Knockdown of either subunit reduces PheRS in Kc cells (Lu et al., 2014). Here, β-PheRS knockdown was used. RNAi knockdown was carried out by directly adding dsRNA to the medium. (C) For analysis of proliferation, Xpd RNAi was used as a control. Cells were harvested on days 1, 2, 3, 4 and 5 after dsRNA treatment. (D-D″) RNAi knockdown reduces the mitotic index. The mitotic index was determined by counting the phospho-Histone H3-positive cells (white dots in D′,D″) and all cells. Over 10,000 cells were counted for each treatment. ****P<0.0001 (unpaired Student’s t-test). Scale bars: 25 μm. (E) Reduced PheRS decreases the cell size of Kc cells. Cell size was determined by measuring the forward scatter

(FSC) in the FACS analysis. raptor RNAi was the positive control, and β-PheRS knockdown showed a similar cell size distribution. Disease Models & Mechanisms

2 RESEARCH ARTICLE Disease Models & Mechanisms (2021) 14, dmm048132. doi:10.1242/dmm.048132 the following days. Compared to the controls, cells treated with levels (Kim et al., 2008). The Rag complex was identified as a nutrient β-PheRS RNAi started to show lower cell numbers on day 3, and the sensor in this pathway, and knockdown of RagA (also known as cell count was ∼75% of that of the control on day 5. In Kc cells, RagA-B) prevents the TORC1 complex from sensing the availability knocking down either subunit alone reduced levels of the α-PheRS of amino acids (Kim et al., 2008; Sancak et al., 2008). We therefore and β-PheRS subunit (Lu et al., 2014). It was therefore reassuring used RagA as our control (Fig. 2A,B). In contrast to RagA, knocking that α-PheRS knockdown showed similar results. Because routine down β-PheRS did not prevent amino acid sensing in this assay, and cell viability assays did not point to an increase in dead cells in any phosphorylation of dS6K was still induced to a similar level as in the of the samples upon RNAi treatment, a change in cell numbers control when amino acids were re-added after deprivation (Fig. 2A,B). should reflect a proliferation change. As a positive control, Xpd In this case, it did not matter whether we re-added all amino acids or RNAi was performed and showed the published increase in cell only L-Phe. Although we cannot rule out that the RNAi knockdown proliferation (Chen et al., 2003). The fact that knockdown of Xpd was insufficient to reveal an amino acid-sensing function for PheRS, can speed up cell growth and proliferation not only indicates that the the results seem to suggest that PheRS might not serve as an amino Kc cells were healthy, but also that the PheRS levels are not limiting acid sensor upstream of the TORC1 complex. for growth and proliferation, but can sustain even higher proliferative activity. Non-canonical α-PheRS activity is sufficient to induce Determining the mitotic index upon PheRS knockdown revealed additional M-phase cells that the reduced PheRS levels caused a strong reduction in mitotic Circumstantial evidence suggests that elevated PheRS levels do not cells as indicated by the lower fraction of phospho-Histone 3 (PH3; simply allow higher translational activity to overcome a growth rate mitotic marker)-positive cells. The RNAi treatment reduced the restriction imposed by hypothetically limiting levels of PheRS. mitotic index to 1.7±0.16%, which corresponded to half of the control PheRS is unlikely to be rate limiting for cellular growth because (3.3±0.16%) (Fig. 1D). Similarly, cell size was also affected by animals with only one copy of α-PheRS (α-PheRS/–)orβ-PheRS PheRS knockdown (Fig. 1E). The cell size showed a reduction that (β-PheRS/–) do not show a phenotype (Lu et al., 2014). Furthermore, was similar to the one observed upon knocking down raptor with Kc tissue culture cells can be stimulated to grow more rapidly without RNAi. Raptor is a component of the TORC1 (target of rapamycin stimulating the expression of α-PheRS or β-PheRS (Fig. 1C; Chen complex 1) signaling that regulates cell growth (Kim et al., 2002). et al., 2003). To test whether elevated levels of PheRS can stimulate These experiments showed that α-PheRS and β-PheRS are needed for growth or proliferation, we expressed α-PheRS, β-PheRS and both normal growth and proliferation of cells, organs and entire animals. subunits together in the posterior compartment of wing discs using a This result might reflect the requirement for the enzymatic activity of Gal4 driver under the control of the engrailed (en) promoter. This PheRS in charging the tRNAPhe with its cognate amino acid en-Gal4 drives the expression of the α-PheRS and β-PheRS open phenylalanine, or it might point to a novel, possibly moonlighting reading frames (ORFs) that were cloned behind a UAS promoter and function of PheRS in stimulating growth and proliferation. integrated into a defined landing platform in the fly genome that also contains the α-PheRS and β-PheRS genes. In this assay, the anterior PheRS lacks apparent amino acid sensor activity for TORC1 compartment expresses normal endogenous PheRS levels and serves The TORC1 signaling pathway activates growth and proliferation of as an internal control, and the expression of, for instance, α-PheRS cells depending on the availability of amino acids, growth factors and driven by en-Gal4 increased the level of α-PheRS signal by 80% energy (Laplante and Sabatini, 2012; Wullschleger et al., 2006). In (Fig. S1). Depending on the specificity of the antibody, the actual addition to its canonical function in charging tRNALeu with leucine, increase in α-PheRS levels might be somewhat higher (for details, see the LeuRS serves as the central amino acid sensor in this pathway Fig. S1). When α-PheRS and β-PheRS levels were raised in the (Bonfils et al., 2012; Han et al., 2012). We therefore tested whether posterior wing disc compartment together, the PH3 labeling revealed PheRS might also be involved in nutrient sensing for TORC1 a 40% increase in mitotic cells in the posterior compartment relative signaling in an analogous way. Amino acid deprivation causes a to the anterior one of the same wing disc (Fig. 3). Surprisingly, the downregulation of phosphorylation of dS6K in Kc cells, and same result was obtained when only the levels of the α-PheRS subsequent stimulation with amino acids restores phospho-dS6K subunit were raised (Fig. 3A-D), but not when only the β-PheRS

Fig. 2. PheRS does not act as an amino acid sensor for the TORC1 complex. (A) β-PheRS knockdown cannot block the TORC1 complex from sensing the availability of amino acids. Phospho-S6K was used as a readout of TORC1 signaling. Starvation (−) was performed by depriving cells of amino acids (AA) for 30 min, and stimulation (+) was performed by re-adding amino acids for 30 min after starvation. The control was a mock RNAi, RagA RNAi is known to block the sensing of amino acids. Phospho-S6K levels were quantified relative to the Actin levels in the same extract. (B) β-PheRS knockdown did not block the TORC1 complex from sensing the availability of L-Phe. The same experiment as in A was performed, but using L-Phe and L-Glu for stimulation. L-Glu was reported to be necessary for amino acid transport (Nicklin et al., 2009). Levels of phospho-S6K were quantified relative to the Actin levels in the same extract. Disease Models & Mechanisms

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Fig. 3. Elevated α-PheRS promotes the appearance of mitotic cells without β-PheRS and without stimulating translation. (A-C‴) Wing disc phenotypes induced by the overexpression of α-PheRS or α- PheRSCys. en-Gal4 was used to drive transgene expression in the posterior compartment of developing wing discs [en-Gal4/+;UAS-GFP/UAS-α- PheRS(Cys)]. Mitotic cells were visualized with anti-phospho-Histone H3 (PH3) antibodies. A, anterior compartment; P, posterior compartment. Scale bars: 29 μm. (D) Mitotic (PH3-positive cells) cells in the posterior (P) compartment increased 40% relative to the anterior (A) one when both α-PheRS and β-PheRS or only the α-PheRS subunit alone were raised. n=10, **P<0.01, ***P<0.001; ns, not significant (paired Student’s t-test). (E) Whereas increasing the expression of α-PheRS and β-PheRS together showed increased β-PheRS levels, elevating the α-PheRS subunit alone did not affect the β-PheRS subunit levels. (F-G″) Protein synthesis did not increase upon overexpression of α-PheRS or α-PheRS and β-PheRS together. en-Gal4 was used to drive the overexpression of the transgenes in the posterior compartment of wing discs. dMyc was used as a positive control (en-Gal4/UAS-Myc:: MYC;UAS-GFP/+). Scale bars: 29 μm. (H) Protein synthesis was measured by the mean intensity of the puromycin (PMY) signal labeling the nascent polypeptides. n=15, ****P<0.0001; ns, not significant (paired Student’s t-test).

subunit levels were raised (Fig. 3D). In addition, raising the α-PheRS type α-PheRS subunits individually or together with β-PheRS subunit alone did not affect the β-PheRS subunit levels (Fig. 3C-C‴, subunits in Escherichia coli, His tag purified them and performed E). Furthermore, aminoacylation requires both PheRS subunits to aminoacylation assays with the appropriate amount of recombinant ligate Phe to the tRNAPhe (Fig. 4A). The fact that elevated α-PheRS soluble proteins that were normalize to bovine serum albumin (BSA) levels alone are sufficient to raise the mitotic index is therefore standards (Fig. 4B). The clear band in Fig. 4B showed that the Cys another strong indication that α-PheRS possesses a proliferative mutation did not affect the solubility of the recombinant proteins. activity that is unlikely to be mediated by increased tRNAPhe α-PheRSCys together with wild-type β-PheRS produced the same aminoacylation. background signal as the α-PheRS subunit alone, and this signal was To test directly whether α-PheRS can increase proliferation clearly lower than the one obtained with wild-type α-PheRS plus without stimulating translation, we made a mutant version of β-PheRS (Fig. 4A). The Drosophila gene encoding the cytoplasmic α-PheRS in which Tyr412 and Phe438 are replaced by cysteine α-PheRS subunit is located on the X chromosome. A P-element (Cys). These substitutions are predicted to block the entrance into the insertion into the 5′-untranslated region of the α-PheRS transcript phenylalanine binding pocket, preventing binding of Phe and (α-PheRSG2060) causes recessive lethality that can be rescued by a aminoacylation of tRNAPhe by the mutant PheRSCys (Finarov et al., genomic copy of wild-type α-PheRS (gα-PheRS)thatsupports 2010). To test whether the PheRSCys substitution indeed reduces the aminoacylation (Lu et al., 2014). A genomic copy of the Cys Cys aminoacylation activity of PheRS, we expressed mutant and wild- aminoacylation-defective α-PheRS , gα-PheRS , did not rescue Disease Models & Mechanisms

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Fig. 4. The α-PheRSCys mutant does not support aminoacylation in vitro. The aminoacylation assay was performed with the mixture of the recombinant protein (α-PheRS or α-PheRSCys and β-PheRS) simultaneously expressed in E. coli. tRNAPhe from yeast was aminoacylated with [3H] phenylalanine. (A) The [3H] phenylalanine decays were counted in a scintillation counter. In this assay, wild-type α-PheRS+β-PheRS subunits together gave a rise in counts per minute (CPM) of up to 47,417, and α-PheRSCys+β-PheRS produced less than 654 CPM. α-PheRS or α-PheRSCys alone produced 210 and 191 CPM, respectively. (B) Purified recombinant protein and protein complexes were controlled and quantified on Coomassie Blue- stained gels using bovine serum albumin (BSA) as a standard. The clear bands indicate that the Cys mutation did not affect the solubility of the recombinant proteins.

the lethality of the α-PheRSG2060 mutant, indicating that the Cys The non-canonical activity emanating from α-PheRS or mutant is indeed not functional in aminoacylation in vivo in α-PheRSCys is capable of inducing more cells to be in mitosis. Drosophila. Despite this apparent lack of aminoacylation activity, Such a phenotype is likely to come about by specifically slowing expressing a transgenic copy of α-PheRSCys in the posterior down progression through M-phase, causing higher numbers of compartment of the wing disc with the en-Gal4 driver caused a cells to remain in the PH3-positive state. Alternatively, the activity 67% increase in the number of mitotic cells in the above assay might either promote proliferation of mitotic cells, or induce (Fig. 3D). The fact that the mutant α-PheRSCys version caused an proliferation in non-cycling cells. increase in mitotic cells at least as strongly as the wild-type α-PheRS expressed in the same way, together with the fact that β-PheRS α-PheRS levels accelerate growth and proliferation overexpression was not needed for this effect, strongly suggests that To test the effects of PheRS levels on growth and proliferation directly the increased mitotic index is promoted without increasing the and in an additional cell type, we set up ‘mosaic analysis with canonical function of PheRS. repressible cell marker’ (MARCM; Wu and Luo, 2006) assays in the We also tested directly whether elevated expression of wild-type ovarian follicle cells. Twin spot clones were generated with one clone α-PheRS and expression of α-PheRS and β-PheRS together are expressing elevated levels of PheRS and the GFP marker, and its twin indeed unable to cause elevated translation as we expected. For this, clone expressing normal endogenous levels of PheRS and serving as we analyzed protein synthesis activity in the two wing compartments an internal control (Fig. 5A). The results of this experiment showed by puromycin (PMY) staining using the ribopuromycylation method that clonally elevated levels of both subunits of PheRS accelerated cell (RPM) (Deliu et al., 2017). Testing this method, we first expressed proliferation, on average, by 32% (Fig. 5B). In contrast, clonal elevated elevated levels of the transcription factor dMyc (also known as Myc) expression of GFP with only the β-PheRS subunit or with GlyRS (also in the posterior wing disc compartment. This positive control led to known as GARS) did not significantly promote clonal expansion increased protein synthesis activity and anti-PMY signal in the dMyc- (Fig. 5B). This confirms that the stimulation of proliferation is specific overexpressing posterior compartment relative to the anterior for PheRS and not a general role of aaRSs. Interestingly, clonal compartment of the same discs. In contrast, neither en-Gal4-driven elevated expression of GFP with the α-PheRS subunit alone also expression of α-PheRS alone nor combined expression with β-PheRS stimulated cell proliferation autonomously by 30% (Fig. 5B), and, increased the puromycin labeling in the posterior compartment intriguingly, this was very close to the 32% increase calculated for the (Fig. 3F-H). The combined results therefore demonstrate clone overexpressing both PheRS subunits (Fig. 5B). Remarkably, the unambiguously that elevated α-PheRS levels cause additional cells increase in number of mitotic cells observed upon α-PheRS to be in mitosis through a non-canonical mechanism that does not overexpression in the posterior compartment of the larval wing discs involve a general increase in translation. (Fig. 3C) was in a comparable range to the proliferation increase in the We also considered the remote possibility that the elevated follicle cell assay (Fig. 5B). These results therefore suggest that α- expression of only one PheRS subunit might alter the ratio of Phe- PheRS levels promote cell proliferation and that α-PheRS levels have tRNAPhe/tRNAPhe (charged tRNAPhe to uncharged tRNAPhe)andthat this activity in different tissues. Interestingly, the tub-Gal4 driver used this might affect the translatability of specific mRNAs with a very high in this experiment to produce the elevated α-PheRS levels was found or a very low frequency of Phe codons. We therefore used a proteomics to cause only a 57% increase in α-PheRS levels in L1 larvae when used approach to study the effect of overexpressing α-PheRS with the strong to solely express this subunit (Fig. S2C). ubiquitous tub-Gal4 driver. We then analyzed the expression of the Similarly, clonal elevated expression of α-PheRSCys alone and Drosophila proteome and correlated it to the Phe frequency in these α-PheRSCys together with β-PheRS (PheRSCys) stimulated cell proteins (Fig. S2A,B). The results showed that there was no, or only a proliferation in the follicle cell twin spot experiment by 28% and minor, negative correlation (−0.0029 using the linear and −0.030 with 25%, respectively (Fig. 2B), again confirming that this effect does the log2 expression data). In conclusion, we could not find evidence not depend on increased canonical PheRS activity. that the unbalanced expression of only one PheRS subunit markedly We also measured the clone size of twin spot clones to find out changes the efficiency of reading Phe codons. whether the elevated PheRS could enhance cell growth. This was Disease Models & Mechanisms

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Fig. 5. α-PheRS overexpression promotes follicle cell proliferation independent of tRNAPhe aminoacylation. Clonal analysis (twin spot) experiment of the effect of overexpression of the PheRS subunits in follicle cells by the MARCM technique [hspFLP/+;Act-Gal4/+;neoFRT82B,tub-Gal80/neoFRT82B,UAS-α- PheRS(Cys)]. After inducing mitotic recombination by expressing the FLPase under the control of the heat-shock (hs) promoter at 37°C for 40 min, a recombining cell will divide and give rise to two proliferating clones. (A) One clone (red) overexpresses PheRS; its twin clone (GFP− signal) does not and expresses normal levels (internal control, outlined with yellow). Scale bar: 29 μm. (B) Three days after inducing the recombination, the average number of cell divisions was calculated for each clone and compared to its twin spot to obtain the proliferation increase (%). (C) Three days after inducing the recombination, the average clone size was calculated for each clone and compared to its twin spot to obtain the clone size increase (%). (D) Three days after inducing the recombination, the average size of a single cell in each clone was calculated and compared to its twin spot to obtain the cell size increase (%). n=30, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant (one-way ANOVA). accompanied by results on cell proliferation that clonally elevated function in stimulating cellular growth and proliferation. Because levels of both subunits of PheRS or single α-PheRS subunit levels of α-PheRS are elevated in many tumor cells compared to augmented clone size by 139% and 102%, respectively, whereas their healthy counterparts, and because a positive correlation elevated levels of single β-PheRS subunit or GlyRS did not result in between these levels and tumorigenic events had been noted some significant increase in clone size (Fig. 5C). We also observed that time ago (Sen et al., 1997), it was important to find out whether the size of single cells in twin spot clones remained unchanged elevated PheRS levels are a mere consequence of the high metabolic (Fig. 5D), whereas the total clone sizes increased according to the activity of the tumor cells or whether they might also contribute to increase in cell number per clone (Fig. 5B,C), indicating that overproliferation of tumor cells. Here, we showed that α-PheRS has cell size control was not affected. These results therefore suggest the potential to promote growth and proliferation and that it can do that α-PheRS levels promote cell growth and proliferation and that this independent of its aminoacylation activity, i.e. through a non- α-PheRS levels have this activity in different tissues. canonical or moonlighting activity. We found unambiguous Alternatively, elevated α-PheRSCys alone and α-PheRSCys together evidence for the non-canonical nature of this proliferative activity with β-PheRS (PheRSCys) stimulated cell proliferation in the follicle and also showed that general translation is not elevated when this cell twin spot experiment by 28% and 25%, respectively (Fig. 5B,C). phenotype is induced by elevated α-PheRS or even the entire PheRS The clonal sizes also expanded by 99% and 86%, respectively, by protein (Fig. 3D-D″). Aminoacylation of tRNAPhe requires both Cys Cys accelerating levels of α-PheRS alone and α-PheRS together subunits to form the tetrameric protein α2β2-PheRS that can with β-PheRS. These observations again confirmed that this effect on aminoacylate tRNAPhe (Fig. 4), and overexpression of an cell growth and proliferation does not depend on increased canonical aminoacylation-dead α-PheRSCys mutant subunit alone (without PheRS activity. simultaneous overexpression of the β-PheRS subunit) increased the cell numbers in the follicle cell clones as much as the wild-type gene DISCUSSION expressed in the same way did (Fig. 4 and Fig. 5B). The described Our work revealed that PheRS not only charges tRNAs with their non-canonical functions of α-PheRS are not only independent of the cognate amino acid Phe, but that it also performs a moonlighting aminoacylation activity, they also do not reflect a function in Disease Models & Mechanisms

6 RESEARCH ARTICLE Disease Models & Mechanisms (2021) 14, dmm048132. doi:10.1242/dmm.048132 sensing the availability of its enzymatic substrate, Phe, for the major In recent years, it has become increasingly evident that many, if not growth controller, the TOR kinase, like the aaRS members TrpRS or most, proteins have evolved to carry out not only one, but two or LeuRS (Fig. 2) (Adam et al., 2018; Bonfils et al., 2012; Han et al., more functions, providing interesting challenges in determining 2012). which of their activities are important for the specific function of a The notion that the α-PheRS subunit can be stable (Fig. 3A″)and gene (Dolde et al., 2014). function independently of the β-subunit (Fig. 3C-C‴,E) was Improper expression of PheRS was suspected long ago to surprising because previous results showed that the two subunits promote carcinogenesis, but until now the mechanisms behind this were dependent on the presence of the other subunit for their stability effect remained unknown (Sen et al., 1997). Elevated mRNA levels (Antonellis et al., 2018; Lu et al., 2014; Xu et al., 2018). Our results of the human α-PheRS, FARSA, during the development of myeloid now show that this requirement does not apply to all cell types. In leukemia correlate with tumorigenic events. The GENT2 database mitotically active follicle cells, the posterior compartment of the wing published in 2019 also describes strong positive correlations disc and possibly other cells, elevated expression of the α-PheRS between PheRS subunit mRNA levels and tumorigenic events in subunit alone results in higher levels of α-PheRS accumulation and several tissues and cancers (Barker et al., 2009; Park et al., 2019). this produced a strong phenotype. This suggests that the α-PheRS and Interestingly, and consistent with our results, not all tumors that β-PheRS subunits function together in every cell to aminoacylate displayed elevated PheRS levels showed elevated levels of α-PheRS tRNAPhe, but, in addition, the α-subunit can be stable in specific cell and β-PheRS mRNA. For instance, brain, ovary, endometrium and types that appear to have retained their mitotic potential. In these cells, bladder tumors displayed only elevated α-PheRS mRNA levels, α-PheRS assumes a novel function in promoting cell growth and whereas colon, breast, lung and liver tumors showed elevated levels proliferation. This would then suggest that many differentiating cell of mRNAs for both subunits. Because elevated levels of α-PheRS or types start to put a system in place that prevents α-PheRS α-PheRSCys alone can elicit mitotic activity, growth and accumulation at high levels. Such a mechanism could then proliferation, our results suggest that the excessive PheRS (FARS) contribute to reducing the proliferative activity of differentiated cells. levels in tumor tissues might be able to produce such proliferative If α-PheRS does not act as an amino acid sensor for TORC1, how signals independent of whether they also produce elevated levels of could it perform this function in growth and proliferation? The β-PheRS. Modeling the effect of elevated α-PheRS levels in report that aaRSs can modify other proteins on Lys side chains with Drosophila, we found that elevated levels support growth and the amino acid they usually attach to the tRNA caused us initially to proliferation and lead to an increase in mitotic cells in different cell investigate this possibility (He et al., 2018). However, our results types. In follicle cells, more cells were produced in clones that the enzymatic activity of PheRS is not required for this effect expressing more α-PheRS compared to wild-type clones. In wing strongly argued against such a pathway. Trying to find possible discs, more mitotic cells were detected in most areas with higher downstream targets of α-PheRS, we went through the list of proteins levels of α-PheRS. This indicates that elevated α-PheRS levels can for which levels significantly increased or decreased in response to indeed be a risk factor for tumor formation in several different the elevated expression of this subunit (Table S1). For only one of tissues. these proteins, Psi, a role in growth and proliferation had been described (Guo et al., 2016). However, this protein becomes MATERIALS AND METHODS significantly underexpressed by elevated α-PheRS levels and this Fly genetics and husbandry would be predicted to cause reduced growth and proliferation. This, All Drosophila melanogaster fly stocks were kept for long-term storage at like many other changes in the expression pattern, is therefore more 18°C in glass or plastic vials on standard food with day/night (12 h/12 h) easily explained by a more indirect effect, like the consequence of light cycles. All experiments were performed at 25°C with female animals increased growth and proliferation (Table S1). Interestingly, the 3D unless specifically mentioned. A UAS-GFP element was added in structure of monomeric α-PheRS in the Swiss-Model online tool experiments that tested for rescue and involved Gal4-mediated expression (Biozentrum, University of Basel; https://swissmodel.expasy.org/ of the rescue gene. This construct served to even out the number of UAS repository/uniprot/Q9W3J5) revealed that the α-PheRS consists of sites in each Gal4-expressing cell. Origins of all stocks are provided in Table S2. two distinct parts (Finarov et al., 2009): the catalytic core with aminoacylation function and, separated by a linker, another part with DNA-binding domains (DBDs) and unknown function DNA cloning and generation of transgenic flies Sequence information was obtained from FlyBase. All mutations and the (Finarov et al., 2010). Furthermore, the DBDs contain a predicted α nuclear localization sequence (NLS) at position 159 addition of the Myc tag to the N-terminus of -PheRS were made by following the procedure of the QuickChange® Site-Directed Mutagenesis (ADFKKRKLLQE) (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_ Kit (Stratagene). The genomic α-PheRS rescue construct (Myc::α-PheRS) Mapper_form.cgi). This region could be a candidate for the one codes for the entire coding region and for an additional Myc tag at the that performs the non-canonical function of α-PheRS, and its N-terminal end. In addition, it contains ∼1 kb of up- and downstream structure might suggest that its activity involves entering the nucleus sequences and it was cloned into the pw+SNattB transformation vector and binding to DNA, or possibly RNA, to stimulate growth and (Koch et al., 2009; Lu et al., 2014). The α-PheRS and β-PheRS cDNAs proliferation. were obtained by RT-PCR from mRNA isolated from 4- to 8-day-old OreR PheRS is not the only aaRS family member with roles beyond flies (Lu et al., 2014). The Tyr412Cys and Phe438Cys mutations in the α- charging tRNAs (Dolde et al., 2014; Guo et al., 2010; Lu et al., PheRS sequence were created by site-directed mutagenesis. Like the wild- 2015; Pang et al., 2014). For instance, MetRS/MRS is capable of type cDNA, they were cloned into the pUASTattB transformation vector to α α Cys stimulating ribosomal RNA (rRNA) synthesis (Ko et al., 2000), generate the pUAS- -PheRS and pUAS- -PheRS . Before injecting these constructs into fly embryos, all plasmids were verified by sequencing GlnRS/QRS can block the kinase activity of apoptosis signal- (Microsynth AG, Switzerland). Table S4 lists the primers used to make the regulating kinase 1 (ASK1) (Ko et al., 2001), and a proteolytically mutants and constructs, and the primers used to confirm their sequence. processed form of YARS/TyrRS acts as a cytokine (Casas-Tintó Transgenic flies were generated by applying the φ C31-based integration et al., 2015; Greenberg et al., 2008). aaRSs are, however, not the system with the stock (y w att2A[vas-φ]; +; attP-86F)(Bischofetal., only protein family that evolved to carry out more than one function. 2007). Disease Models & Mechanisms

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Western blotting dried, they were immersed in PPO Toluol (Sigma-Aldrich) solution in Protein was extracted from tissues, whole larvae or flies using lysis buffer. plastic bottles and the radioactivity was measured by scintillation counting. Protein lysates were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Blocking was performed for 1 h at room Wing disc dissociation and fluorescence-activated cell sorting temperature (RT) with non-fat dry milk (5%) in Tris-buffered saline with (FACS) analysis Tween (TBST) solution. Blots were probed first with primary antibodies Wandering larvae derived from 2-4 h egg collections were dissected in PBS (diluted in blocking buffer) overnight at 4°C and then with secondary during a maximal time of 30 min. Around 20 wing discs were incubated with antibodies (diluted in TBST) for 1 h at RT. The signal of the secondary gentle agitation at 29°C for ∼2 h in 500 µl 10× trypsin-EDTA supplemented ™ antibody was detected using the detect solution mixture (1:1) (ECL Prime with 50 µl 10× Hank’s balanced salt solution (Sigma-Aldrich) and 10 µl Western Blotting System, GE Healthcare Life Science) and a luminescent Vybrant DyeCycle Ruby stain (Molecular Probes, USA). Dissociated cells detector (Amersham Imager 600, GE Healthcare Life Science). Origins of from wing discs were directly analyzed by FACS-Calibur flow cytometer reagents and recipes for buffers are provided in Tables S2 and S3, (Becton Dickinson, USA). respectively. Drosophila tissue culture cells were harvested and fixed in 70% ethanol and stained with a staining solution containing 1 mg/ml propidium iodide, Immunofluorescent staining and confocal microscopy 0.1% Triton X-100 and 10 mg/ml RNase A. The cells were then subjected to Dissections were performed in 1× PBS on ice and tissue collected within a FACS-Calibur cytometry and data were analyzed with the FlowJo software. maximum of 1 h. Fixation was performed with 4% paraformaldehyde (PFA) in 0.2% phosphate buffered saline with Tween (PBST) for 30 min at RT Drosophila cell culture and RNAi treatment (wing discs, ovaries). Then the samples were blocked overnight with Drosophila Kc cells were incubated at 25°C in Schneider’s Drosophila blocking buffer at 4°C. Primary antibodies (diluted in blocking buffer) were medium supplemented with 10% heat-inactivated FCS and 50 µg/ml penicillin/ incubated with the samples for 8 h at RT. The samples were rinsed three streptomycin. To induce RNAi knockdown in Drosophila cells, dsRNA times and washed three times (20 min/wash) with PBST. Secondary treatment was performed (Clemens et al., 2000). dsRNAs around 500 bp in antibodies (diluted in PBST) were incubated overnight at 4°C. The samples length were generated with the RNAMaxx™ High Yield Transcription Kit were then rinsed three times and washed two times (20 min/wash) with (Agilent, USA). Cells were diluted to a concentration of 106 cells/ml in serum- PBST. Hoechst 33258 (2.5 μg/ml) was added in PBST before the last free medium, and dsRNA was added directly to the medium at a concentration washing step and the samples were mounted with Aqua/Poly Mount of 15 µg/ml. The cells were incubated for 1 h followed by addition of medium solution (Polysciences, USA). Origins and dilutions of all antibodies are containing FCS. Then the cells were kept in the incubator and were harvested at provided in Table S2. different time points 1-5 days after dsRNA treatment.

Protein synthesis measurements using RPM Clonal assay and twin spot data analysis For puromycin labeling experiments, tissues were dissected in Schneider’s For twin spot tests, we used the MARCM system. Twin spots were generated β insect medium (Sigma-Aldrich, USA) supplemented with 10% fetal calf with the progenitor genotype hs-flp; tub-Gal4/UAS- -PheRS; FRT82B, α (Cys) serum (FCS; Sigma-Aldrich) at 25°C. They were then incubated with ubiGFP, UAS- -PheRS /FRT82B Tub-Gal80. In twin spots, the internal Schneider’s insect medium containing puromycin (5 μg/ml, Sigma-Aldrich) control clone was GFP-minus and the twin sister clone produced a red signal by and cycloheximine (100 µg/ml, Sigma-Aldrich) for 2 h at RT. Then the the antibody against the overexpressed protein. We induced the hs-FLP, samples were fixed with 4% PFA in PBST 0.2% at RT and blocked FRT82B system at 37°C for 1 h on the third day post-eclosure and dissected the overnight with blocking buffer at 4°C. Primary anti-Puromycin antibody animals 3 days post-induction. Confocal imaging detected non-green clones (diluted in PBST) was incubated with the samples for 8 h at RT. The (without ubiGFP) and red clones (stained with anti-Myc antibody) (Fig. 5A). samples were rinsed three times and washed three times (20 min/wash) with In twin spots, cell numbers per clone were counted and the numbers of PBST. Secondary antibodies (diluted in PBST) were incubated overnight at cell divisions per clone were calculated as log2(cell numbers per clone). This 4°C. The samples were then rinsed three times and washed two times represents the logarithm of the cell numbers per clone to the base 2. The (20 min/wash) with PBST. Hoechst 33258 (2.5 μg/ml) was added in PBST increase in cell proliferation (%) was analyzed by comparing the number of before the last washing step and the samples were mounted with Aqua/Poly cell divisions of the clone pairs in the same twin spot. The clone sizes were Mount solution (Polysciences). See Table S2 for antibody details. measured by FIJI software, and the increase in clone size was analyzed by comparing the clone size in the same twin spot.

In vitro aminoacylation assay Image acquisition and processing Recombinant α-PheRS and β-PheRS proteins were co-expressed in the E. Imaging was carried out with a Leica SP8 confocal laser scanning coli strain Rosetta (Novagen) and then purified (Moor et al., 2002). For this, microscope equipped with a 405 nm diode laser, a 458, 476, 488, 496 and the α-PheRS or α-PheRSCys mutant cDNAs were cloned with His tags at the 514 nm Argon laser, a 561 nm diode-pumped solid-state laser and a 633 nm N-terminal end into the pET-28a plasmid expression vector (Novagen). HeNe laser. Images were obtained with 20× dry and 63× oil-immersion Wild-type β-PheRS cDNAs were cloned into the pET LIC (2A-T) plasmid objectives and 1024×1024 pixel format. Images were acquired using LAS X (Addgene #29665). Then, His-α-PheRS or the His-α-PheRSCys mutant and software. The images of the entire gut were obtained by imaging at the β-PheRS were co-expressed in the E. coli strain Rosetta with standard size and then merging maximal projections of z-stacks with the isopropylthiogalactoside (1 mM) induction at 25°C for 6 h. Proteins were Tiles Scan tool. Fluorescence intensity was determined using FIJI software. purified with Ni-NTA affinity resin (Qiagen). The aminoacylation assay protocol from Lu et al. (2014) was then followed, with the modification that the Whatman filter paper discs were soaked in phenylalanine solution for 1 h Quantification and statistical analysis [30 mg/ml in 5% trichloroacetic acid (TCA)] to reduce the background. This For quantifications of all experiments, n represents the number of assay was performed at 25°C in a 100-μl reaction mixture containing 50 mM independent biological samples analyzed (the number of wing discs, the Tris-HCl pH 7.5, 10 mM MgCl , 4 mM ATP, 5 mM β-mercaptoethanol, number of twin spots), error bars represent s.d. Statistical significance was 2 ’ 100 μg/ml BSA, 3 U/ml E. coli carrier tRNA, 5 μM[3H]-amino acid (L- determined using Student s t-test or ANOVA as noted in the figure legends. Phe) and 1 μM tRNAPhe from brewer’s yeast (Sigma-Aldrich). In each experiment, a 15-μl aliquot was removed at four different incubation time Acknowledgements We thank Hugo Stocker, Albena Jordanova, Erik Storkebaum and the Bloomington points, spotted on the Phe-treated Whatman filter paper discs and washed Drosophila Stock Center for fly stocks. We also acknowledge the support of the three times with ice-cold 5% TCA and once with ice-cold ethanol. A blank imaging and proteomics facility of the University of Bern, and we are grateful to Mark paper disc without spotting and another with spotting of the enzyme-free Safro for suggesting mutations that disrupt the phenylalanine binding site of reaction were used for detecting background signals. After filter discs were α-PheRS. We further thank Jonathan Huot for his valuable advice on the Disease Models & Mechanisms

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H., Kim, H. K., Ha, S. H., M.T.H., J.L., D.B., B.S.; Investigation: M.T.H., J.L., D.B.; Writing - original draft: Ryu, S. H. and Kim, S. (2012). Leucyl-tRNA synthetase is an intracellular leucine M.T.H., J.L., B.S.; Project administration: B.S. sensor for the mTORC1-signaling pathway. Cell 149, 410-424. doi:10.1016/j.cell. 2012.02.044 Funding He, X.-D., Gong, W., Zhang, J.-N., Nie, J., Yao, C.-F., Guo, F.-S., Lin, Y., Wu, X.-H., This work was supported by Novartis Stiftung für Medizinisch-Biologische Li, F., Li, J. et al. (2018). Sensing and transmitting intracellular amino acid signals Forschung (18A050), Schweizerischer Nationalfonds zur Förderung der through reversible lysine aminoacylations. Cell Metab. 27, 151-166.e6. doi:10. Wissenschaftlichen Forschung (project grant 31003A_173188) and the Universität 1016/j.cmet.2017.10.015 Bern to B.S. J.L. was also supported by the Schweizerischer Nationalfonds zur Kim, D.-H., Sarbassov, D. D., Ali, S. M., King, J. E., Latek, R. 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