Protein of Interest Synthesis Using Non-formylated Methionine−Escherichia coli initiator tRNA in HeLa cells

doi:10.21203/rs.3.rs-1602404/v1

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Protein of Interest Synthesis Using Non-formylated Methionine−Escherichia coli initiator tRNA in HeLa cells

https://doi.org/10.21203/rs.3.rs-1602404/v1

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In eukaryotic cells and Escherichia coli, protein synthesis is initiated through non-formylated and formylated methionine-conjugated initiator tRNA (Met-tRNAi and fMet-tRNAi, respectively).

Studies have shown that protein of interest (POI) reduced EGFP mRNA expression in human Met-tRNAi/human methionyl-tRNA synthetase (HMRS) pairs under methionine-deficient conditions. Intracellular L-Met depletion can result in the inhibition of endogenously non-formylated human Met-tRNAi/HMRS in HeLa cells.

We investigated the effect of non-formylated Met-conjugated E. coli initiator tRNA (E. coli Met-tRNAi) on POI synthesis in HeLa cells under intracellular L-Met levels. The expression of GFP—a model protein—in L-methionine-deficient HeLa cells, in which endogenous tRNAi was replaced with two kinds of E. coli tRNAi, E. coli Met-tRNAi/E. coli methionyl-tRNA synthetase (EcMRS) and E. coli Met-tRNAi/HMRS, was analyzed.

The results indicated that E. coli Met-tRNAi can offset human Met-tRNAi, and E. coli methionyl tRNA synthetase (EcMRS) can drive the expression of EGFP mRNA.

The non-formylated E. coli Met-tRNAi/EcMRS pairs reduced the expression of EGFP mRNA, resulting in reduced HeLa cell survival.

Our results provide new insights into the potential use of E. coli Met-tRNAi as novel therapeutics that can control POI synthesis initiation. The E. coli Met-tRNAi/MRS pair has the potential to fine-tune protein synthesis in mammalian cells.

tRNA

E. coli initiator tRNA

translation initiation

non-formylated methionine

formylated methionine

E. coli methionyl-tRNA synthetase

Protein engineering technologies based on cellular systems (e.g., mammalian, microbial [bacteria and yeast], insect, plant, and transgenic animal systems) have been developed on an industrial scale [1]. Protein of interest (POI) expression is introduced for regulating protein folding, post-translational modification, and product assembly, which are important biological activities in mammalian cells [2–5]. The introduction of POIs based on large-scale recombinant protein production using gene or promoter transcriptional tools/resources and human codons has been further expanded for high-efficiency protein expression [6–9].

As one of the most complex biological processes involving cofactors and enzymes in cells, the initiation of protein synthesis can result in various initiator transfer RNA (tRNAi) motifs [10, 11]. tRNAis carry essential structural elements, including anticodon loops, variable loops, acceptor stems, dihydrouridine loops, and T ψ (psi) C loops, for tRNA targeting of the ribosomal P site in newly synthesized proteins [12, 13]. The effect of the stability of the initial P-site tRNA and mRNA complexes on translation frame movement has commonly been observed using the ribosome frame mechanism [14–16]. Translational frameshifts should allow for stabilization of the P-site codon/anticodon pairs established in the initial and novel translation frames of the initial non-formylated tRNA-mRNA machinery [17, 18]. Folding a single polynucleotide chain to form a double-helix stem-and-loop region is an essential function of this structure (Fig. 1), which is typical of all tRNAs.

In Escherichia coli, protein synthesis is generally initiated using a formylated methionine tRNAi (fMet-tRNAi). However, in eukaryotic organelles such as mitochondria and chloroplasts, protein synthesis is initiated by non-formylated Met-tRNAi. Studies of the E. coli system have revealed that fMet-tRNAi is charged by E. coli methionyl-tRNA synthetase (EcMRS) [19]. A novel strategy for the cell-selective metabolic labeling of proteins in complex cellular mixtures was reported in 2018 using EcMRS (referred to as NLL-EcMetRS) [20, 21]. The heterologous expression of human tRNAi using mutant EcMRS was reported for incorporating azidonorleucine into proteins produced in HEK293 cells [19].

Furthermore, tRNA modification promotes the efficiency and accuracy of translation, in turn influencing the fidelity of protein synthesis, recognition by elongation aminoacyl-tRNA synthetases (aaRS), and efficiency of translation reading frames [22, 23]. Considering that many tRNAs possess unique features, we questioned whether tRNAs are sequestered exclusively for the initiation of protein synthesis or are associated with the role of tRNAis in protein synthesis, as shown in Supplementary Figure S1. It is highly likely that one modified nucleotide is present in the tRNA, suggesting that the process of maintaining the translational reading frame evolved early in the emergence of new phylogenetic domains [24].

Most tRNA modifications can also have specific functions with respect to the aaRS reaction. Previous studies revealed that fMet-tRNAi-dependent mistranslation could lead to protein degradation, an essential function in cellular homeostasis, and the destruction of anomalous proteins [25–27].

Aminoacyl tRNA has high substrate selectivity and faithful synthesis by editing non-cognate products via selective aaRS activity [28]. Thus, the pairing of cognate tRNAs in aaRS can be used to determine the fidelity of protein translation. The low specificity achieved by misaminoacylation can result in mistranslation, which can be lethal or contribute to the development of pathologies in mammalian cells [29]. However, the acceptor end of tRNAi/aaRS pair complex formation remains controversial [30, 31].

In addition, aaRS can explain the association of amino acids and genetic codes by catalyzing the correct aminoacyl ester-linked tRNA process through a two-step reaction. Methionine-containing amino acids play a crucial role as substrates for the initiation of protein synthesis in mammalian cells [12, 32], thereby regulating cellular metabolism and the proliferation rate, which is much more complex than protein synthesis. The methionine-dependent Hoffman effect has also been reported for cancer cells, which rely on exogenous methionine [33]. The role of the Met-tRNAi complex is proposed to regulate the translation of key genes involved in tumorigenesis owing to its unique function in translation initiation. Although tRNA and aminoacyl-tRNA synthesizer (ARS), which are involved in protein translation, are considered “housekeeping” molecules without functional activity [34], many studies have reported that ARS is also involved in various physiological and pathological processes, particularly tumorigenesis [34–36].

In 2017, we reported a POI detection strategy wherein the fluorescent-labeled N-terminal was conjugated to Met-tRNAi (fluorescent Met-tRNAi) to validate the occurrence of protein synthesis [37]. During synthesis, the fluorescent Met-tRNAi with a purified Cy5-Met process allowed the mRNA encoding the human Met-tRNAi complex to synthesize the fluorescent-labeled POI in HeLa cells. In this study, we investigated whether E. coli Met-tRNAi is the tRNAi capable of initiating POI synthesis in HeLa cells. We hypothesized that the initiation of protein synthesis in eukaryotic cells is promoted via translation events involving E. coli Met-tRNAi/EcMRS pairs [38]. The results demonstrated the association between POI synthesis and cell viability via the initiation of the translational reading frame of E. coli Met-tRNAi/EcMRS pairs.

Materials

Aminoacyl-tRNA synthetase from Escherichia coli (cat. no. 3646-10KU) and L-methionine (Met) (cat. no. M9625) were purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, 5-carboxyfluorescein (5-FAM), succinimidyl ester dye was purchased from Anaspec (San Jose, CA, USA) and used to fluorescently label the E. coli initiator methionine tRNA (E. coli tRNAi). tRNA was prepared using RNeasy Mini Kits (Qiagen, Hilden, Germany) and amplified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). HeLa cells, obtained from American Tissue Type Collection (Manassas, VA, USA), were cultured in MEM (without phenol red) purchased from Welgene Inc. (Daegu, Republic of Korea). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA).

In vitro human initiator tRNA and E. coli initiator tRNAs synthesis

The PCR product described above was used as a template for in vitro transcription [39] by T7 RNA polymerase (Promega, Madison, WI, USA) to synthesize the E. coli initiator tRNA (tRNAi) sequences [40, 41] (Fig. 1). The conservation of the AUCG sequence among eukaryotic initiator tRNAs suggests that this sequence plays an essential role in the properties of initiator tRNAs (Figure S1A). The Watson–Crick base pair indicates unique characteristics at the end of the acceptor stem and the presence of a purine acceptor stem of E. coli initiator tRNAs (Figure S1B). Prokaryotic and eukaryotic initiator tRNAs include three guanines and three cysteines within the amino acid-anticodon stem, which form three GC pair repertories.

Modified initiator tRNA methionylation

To prepare human fluorophore-conjugated methionyl initiator tRNA (fluorescent Met-tRNAi) and E. coli fluorescent Met-tRNAi, the in vitro transcribed human initiator tRNAs were aminoacylated with purified fluorophore-conjugated methionine using refined human MRS [42] in the reaction buffer. The fluorophore 5-FAM added to the Met-tRNA(Met) after aminoacylation. The methionylation process was conducted in a reaction mixture containing 30 mM HEPES (pH 7.4), 100 mM potassium acetate, 10 mM magnesium acetate, and 100 mM ATP to produce the methionine-charged initiator tRNA. The E. coli initiator tRNAs were aminoacylated with purified fluorescent-labeled methionine, using purified preparations of E. coli methionyl-tRNA synthetase (MRS) and human MRS. After incubation at 37°C for 10 min, 0.1 volume of 2.5 M NaOAc (pH 4.5) was added to the reaction mixture. E. coli tRNA was then extracted with 10 mM phenol-saturated NaOAc (pH 4.5), ethanol-precipitated, and centrifuged. The fluorescent-labeled methionine E. coli tRNAi in the pellet was dissolved in RNase-free water for subsequent methionylation of the human initiator tRNA.

Isolation of fluorescent-labeled methionine-charged initiator tRNA

The amine reactive group of the N-terminal methionine was conjugated with the fluorophore Cy5 (GE Healthcare, Piscataway, NJ) and 5-FAM dye (Sigma-Aldrich, St Louis, MO, USA). Fluorophore-conjugated methionine was prepared by conjugating the α-amino group of L-methionine with 5-FAM dye. Briefly, 0.1 mg of 5-FAM NHS ester dye was dissolved in 0.1 ml of 62.5 mM sodium tetraborate buffer (pH 8.5). After adding about 1 mg of L-methionine in 62.5 mM sodium tetraborate buffer (pH 8.5, 1.0 mL) at room temperature, the mixture was incubated overnight.

Analysis of L-methionine (L-Met) and fluorophore-conjugated methionine with high-performance liquid chromatography

The in vitro transcription of human tRNAi (73 base pairs) and charged methionine E. coli tRNAi, using T7 RNA polymerase (Promega, Madison, WI, USA), was detected by agarose gel separation and size exclusion chromatography (SEC)–high-performance liquid chromatography-fluorescence detection (SEC-HPLC). The analysis of fluorescent-labeled methionine was purified by reverse phase chromatography using a C18 column (4.6 × 250 mm) on an Agilent HPLC system. Analysis of purification by reverse-phase chromatography showed that the solvent system consisted of solvent A (0.1% trifluoroacetic acid (TFA), 99.5% purity) (Sigma-Aldrich) and solvent B (0.1% TFA in acetonitrile), and was linear against solvent B, with a gradient of 20–100% and a flow rate of 1 mL/min. Purity was confirmed to be > 95%.

Cell culture and enhanced green fluorescent protein (EGFP) transfection via methionine presence in media

MEM medium without phenol red (Welgene Inc., Daegu, South Korea) was cultured in HeLa cells, supplemented with 10% fetal bovine serum (v/v) and 1% penicillin/streptomycin (v/v) in 5% CO₂ at 37°C. The cells were transfected with 2 µL of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at 60–90% confluence using 500 ng plasmid per 2 × 10⁶ cells. The grown cell medium was washed twice with 10× phosphate buffered saline (PBS) at pH 7.4, and once with PBS alone. For EGFP expression, a change to methionine-free MEM (without phenol red) was performed before 3–6 h co-transfections (Welgene Inc., Daegu, Republic of Korea) [35, 37] of EGFP plasmid. E. coli initiator tRNA (< 100 pmol per 2 × 10⁶ cells), plus L-methionine, were performed using Lipofectamine 2000, at 37°C, for 24 h (Fig. 4). HeLa cells were co-transfected with EGFP plasmid and 5-FAM-labeled methionine E. coli initiator tRNA using Lipofectamine 2000. Briefly, the expression of 5-FAM-labeled nascent proteins with initiator tRNA, involved the co-transfection of a mixture of EGFP plasmid and 5-FAM-labeled methionine E. coli initiator tRNA, were co-transfected using Lipofectamine 2000 at 37°C for 24 h.

Construction of expression plasmid (EGFP)

An EGFP gene (720 nucleotide pairs) was obtained by the PCR amplification of a pEGFP-N1 vector (Takara Bio, Ann Arbor, MI, USA) using the following PCR conditions: 95°C for 10 min, 25 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 2 min, followed by 72°C for 5 min. The forward primer was 5’-GGCACAAGCTGGAGTACAAC-3' and the reverse primer was 5’-ATGCCGTTCTTCTGCTTGTC-3'. The PCR product was then cloned into pcDNA3.1+ (Invitrogen), at the KpnΙ and SalΙ sites, using the standard restriction cloning methods.

Flow cytometry for determination of E. coli initiator tRNA activity

The level of EGFP expression was assessed in two different growth stages, namely the logarithmic and stationary stages. EGFP expressed in HeLa cells was harvested by trypsinization, centrifuged at 3000 rpm for 15 min, washed, resuspended at 10⁶ cells/ml in PBS, and stored on ice. Harvested EGFP cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with a 15 mV, 488 nm, air-cooled argon ion laser. The FACS caliber flow cytometry results recorded 50,000 events, and EGFP expression was assessed in Met^‒ or Met⁺ medium. The EGFP fluorescence of each cell was identified and calculated using standard optics. Color compensation was performed to eliminate artifacts due to the overlap of EGFP fluorescence. Analysis of EGFP overexpression was performed using laser-induced fluorescence and a BD FACSVerse flow cytometer. The flow cytometry histograms of EGFP protein expression were determined using EGFP fluorescence. EGFP fluorescence obtained images by flow cytometry were normalized to the same intensity range, with an acquisition time of 2.5 s. Flow cytometric analysis was performed using BD FACSuite software and Flow Jo (Becton, Dickinson & Company, Franklin Lakes, NJ, USA).

qRT-PCR

Total HeLa cell RNA for qRT-PCR was extracted using RNeasy Mini Kits (Qiagen) according to the manufacturer's instructions. Total RNA (0.15 µg) was used to prepare single-stranded cDNA, using Superscript III Reverse Transcriptase (Invitrogen) and an oligo dT primer. Real-time qPCR was performed using 15 ng of cDNA, SYBR Green (LightCycler® 480 SYBR Green I Master), and LightCycler PCR equipment (Roche Diagnostics, Basel, Switzerland). The PCR conditions were as follows: 95°C for 5 min, followed by 40 cycles of 10 s at 95°C for 15 s, 56°C, and 72°C for 20 s. The EGFP forward primer was 5’-GGCACAAGCTGGAGTACAAC-3' and the reverse primer was 5’-ATGCCGTTCTTCTGCTTGTC-3'. All measurements were normalized to GAPDH.

Mass analysis of the modified initiator tRNA

Fluorescent-labeled methionine was purified using human Met-tRNA synthetase preparations. After incubation at 37°C for 10 min, 0.1 volume (2.5 M) (pH 4.5) was added to the reaction mixture. The tRNA was then extracted with phenol saturated with 10 mM NaOAc (pH 4.5), ethanol precipitated, and centrifuged. The fluorescent-labeled methionine initiator tRNA pellet was dissolved in RNase-free water. The in vitro transcription of fluorescent methionine human initiator tRNA via T7 RNA polymerase human initiator tRNA (73 bp) was detected by agarose gel separation and MALDI-TOF/TOF 5800 (AB Sciex, Toronto, Canada) in the linear and positive ion modes [43]. For all experiments, the matrix solution consisted of 3-hydroxypicolinic acid (50 g/L in H₂O) and diammonium citrate (50 g/L in H₂O). With external calibration using sinapinic acid for BSA, the mass accuracy was estimated to be approximately 0 + 1%, or 25 kDa, for tRNAs of this size. An internal calibration was performed.

Fluorescent and biotin labeling Tat protein detection

Tat protein was produced via in vitro translation, complexed with fluorescent-methionine-E. coli initiator tRNA, separated by SDS-PAGE, and identified by staining with Coomassie blue (lower panel). The amount of fluorescent Tat protein expression was separated by SDS-PAGE and visualized. For the freeze-thaw lysis of mammalian cells, HeLa cells were plated on 6-cm dishes (2 × 10⁴/dish) (Nalge Nunc, Rochester, NY, USA) and lysed with FT LYSIS Buffer containing 600 mM KCl, 20 mM Tris-Cl (pH 7.8), and 20% glycerol. The HeLa lysate was centrifuged at 15,000 rpm at 4°C for 15 min, and the clarified supernatant was then loaded onto HisPur™ Ni-NTA Spin Columns (Pierce Biotechnology, Rockford, IL, USA). The composition of the binding and elution reactions was 50 mM sodium phosphate and 300 mM sodium chloride (PBS) without 10 mM imidazole at pH 7.2 and pH 5.8, respectively. The fluorescent-labeled Tat was separated by SDS-PAGE and stained with Coomassie Blue. Fluorescence image analysis of fluorescent biotinylated Tat expression was conducted using an inverted fluorescence microscope (IX71; Olympus, Japan) (10× magnification). The fluorescence images were normalized to the same intensity range: 488 nm and 545 nm laser lines were used for the green filter. The laser was focused on the channel using a 20× objective lens. The fluorescence signals were collected using the same lens and were optically filtered. Green filter set U-MWIBA3 EGFP shift-free (BP460-495 BA510-550) was used to detect green fluorescence.

Cell viability assay

The HeLa cell lines viability status was determimned using a D-Plus™ CCK cell viability assay kit (Dongin LS, Seoul, Korea) in accordance with the manufacturer's protocol. HeLa cells were seeded in 96-well plates that contained 100 µl of 3 × 10³ cells/well. After the seeded cells had been cultured for 24 h at 37°C, the maintained MEM medium was changed between methionine - MEM (without phenol red) and Methionine + MEM (without phenol red) (Welgene Inc., Daegu, South Korea), respectively.

To assess toxicity following E. coli initiator tRNA treatment, the cells were treated with diluent concentrations (0,1,5 and 10 nM) of E. coli initiator tRNA from E.coli initiator tRNA (10 µM/mL) for 24 h.

Following culturing the cells (1×10⁴ cells per ml) for 24h, the methionine-free MEM- or methionine-treated MEM was added with 100 µL of CCK working solution to each well, and the pate contained cells were further incubated for 4 h at 37°C. The absorbance was measured at 450 nm using a SpectraMax i3 Multi-Mode Platform Microplate Detection (Molecular Devices, LLC, Sunnyvale, CA, USA). The OD value of control cells was considered to represent 100% viability[44].

Statistical analysis

All values are expressed as means. Error bars indicate the standard deviation. Graph Adobe CS7 software was used for graphing and statistical analysis.

Engineering-based synthesis of fluorescent-labeled non-formylated methionine-conjugated E. coli initiator tRNA (Met-tRNAi) and schematic protein translation

Figure 1 shows the non-formylated methionine process for protein synthesis in eukaryotic cells using E. coli Met-tRNAi. We first engineered and heterologously expressed fMet-tRNAi, wherein two types of E. coli Met-tRNAi were introduced: E. coli Met-tRNAi/EcMRS and E. coli Met-tRNAi/human methionyl tRNA synthetase (HMRS). Initiator tRNA is typically used for the initiation of protein synthesis [12, 13, 45]. We compared the activity of human Met-tRNAi and E. coli Met-tRNAi by monitoring the expression of EGFP or fluorescent N-terminal-labeled HIV transcription activator protein (Tat) as model proteins. The human Met-tRNAi was used as a positive control for the fluorescence-labeled E. coli Met-tRNAi-transfected cells (Fig. 1). Codons and amino acids were then assigned to individual tRNAs through selective aminoacylation of the tRNAs using aaRSs [46]. As shown in Supplementary Figure S1 [47, 48], in both prokaryotic and eukaryotic initiators, tRNAs have a common cloverleaf structure and contain all invariant and semi-invariant sequences [47–50].

All eukaryotes and archaebacteria use Met-tRNAi to translate genetic information into a peptide sequence [45]. The tRNAis are used solely for the initiation of protein translation, whereas elongator tRNAs are used for inserting methionine during protein translation [12, 45]. As tRNAi does not bind to elongation factors in the ribosomal A-site, according to previous studies [40, 51], tRNAi imparts a unique structural conformation on the anticodon loop, which may be important for centering on the ribosomal P-site during the initiation of protein translation [12]. Preservation of the sequence is a unique property of eukaryotic tRNAi (Supplementary Figures S1A). The unique characteristics of prokaryotic tRNAi may explain the strong conservation of the Watson–Crick base pair at the end of the receptor stem and the presence of the 11-purine:24-pyrimidine base pair (instead of the 11-pyrimidine:24-purine base pair) (Supplementary Figures S2B). Three consecutive GC pairs, including sequences of three guanines and three cysteines, were formed in the anticodon stem in both prokaryotic and eukaryotic tRNAis (Table 1). The schematic in Fig. 1 summarizes the experiment and compares the activity of human and E. coli tRNAis during the initiation of POI synthesis in mammalian cells.

Table 1. Structural comparison of human initiator tRNAs and E. coli initiator tRNAs

^aAdapted from Drabkin, H. J.et al. (1975).

^bAdapted from B L Seong. et al. (1987).

The fluorescent methionine-conjugated E. coli initiator tRNA mediates the expression of Tat proteins in HeLa cells

To evaluate E. coli Met-tRNAi conjugated with the N-terminal fluorophore 5-FAM (Ex: 492 nm; Em: 512 nm), HeLa cells were extracted from the C-terminal interaction partner fused to the AVI-tagged Tat protein to confirm fluorescent Tat expression (Fig. 2A). To confirm the translation efficiency of the fluorescence-labeled protein using E. coli Met-tRNAi/EcMRS pairs, we prepared a single fluorescent methionine-conjugated E. coli tRNAi with an optimized N-terminal recognition motif, and the C-terminal interaction partner was fused to AviTag. Fluorescent protein imaging was performed using biotin double-labeled Tat on polyethylene streptavidin-coated quartz slides.

Figure 2B presents gel images of the fluorescence-labeled methionine-conjugated E. coli tRNAi-mediated N-terminal labeling of Tat protein. We first investigated whether the fluorescent methionine-conjugated E. coli i tRNAi was involved in the translation of the reading frame using the Tat protein, a factor essential for the transcription of the HIV-1 genome. Fluorescent-labeled human tRNAi-charged endogenous human MRS (lane 2) and E. coli tRNAi-charged EcMRS (lanes 3, 4, and 5), identified via the expression of Tat as a positive control (17 kDa), were analyzed by fluorescence scanning and SDS-PAGE (Fig. 2b). The results showed that E. coli Met-tRNAi/EcMRS pairs migrated in a fluorescent band pattern with fluorescent-labeled Tat protein expression as the positive control. One of the two suggested hypotheses was that the E. coli Met-tRNAi/HMRS pairing with the fluorescent band would result in Tat identification as a 17-kDa band when expressed Tat is dyed with Coomassie brilliant blue staining solution (lanes 1 and 2 in Fig. 2C). HMRS and EcMRS utilization led to the identification of mRNAs involved in Tat expression (Figs. 3 and 4). In contrast, the efficiency of fluorescent protein initiation synthesis by fluorescent-Met-tRNAi/EcMRS was not clearly detected due to the disruption of endogenous protein expression in the Coomassie-stained gel.

These results demonstrated that E. coli Met-tRNAi enables target protein synthesis in live HeLa cells and allows for visualization of the proteins based on tagged fluorescent imaging molecules in response to protein translation mediated by tRNAi/MRS pairs. In addition, fluorescence-tagged Tat protein expression occurred simultaneously with Tat protein expression based on tRNAi translation.

As shown in Fig. 2, fluorescent methionine was chemically synthesized and then purified to confirm the occurrence of protein synthesis [37, 52, 53]. We introduced fluorescence-labeled Tat protein with non-purified fluorescence-labeled Met-conjugated human tRNAi to HeLa lysates. To evaluate the non-purified fluorescent Tat protein obtained using SDS-PAGE, we examined the fluorescent Tat, which was double-tagged with biotin, on streptavidin-coated slides. The initiation of protein synthesis suggested that tRNAis can serve as novel regulators to maintain the translational reading frame.

Validation of an E. coli Met-tRNAi HeLa cell platform in a methionine-deficient medium

The results described above provide experimental evidence of Tat expression using a fluorescence-labeled, methionine-conjugated human tRNAi probe (Fig. 1). EGFP expression was measured in an L-Met–deficient medium, demonstrating the translational recognition of human Met-tRNAi-mediated selective protein expression in HeLa cells [37, 42]. In Met-sufficient growth medium, cells were cultured with all other 19 natural amino acids, whereas fluorescence-labeled methionine was added to the cells during the stationary growth phase. To initiate E. coli Met- tRNAi-mediated selective protein synthesis, we co-transfected an EGFP-expressing plasmid with human Met-tRNAi into HeLa cells. As shown in Fig. 3A, EGFP-expressing cells exhibited lower levels of EGFP mRNA, and the EGFP expression level was reduced in both methionine-free and -sufficient cells.

EGFP mRNA levels determined using RT-qPCR were correlated with the green fluorescence intensity (Fig. 3A, left). Despite the low levels of endogenous tRNAis in the l-Met–free medium, indicating low levels of EGFP expression, this strategy was found to be applicable for using E. coli tRNAi as the positive control (Fig. 3A). The flow cytometry results indicated that EGFP expression gradually increased in the methionine-supplemented medium, as did the level of EGFP. EGFP was highly expressed in Met-supplemented media (GFP population 1.64, dark violet), whereas there was clear quantitative separation of EGFP mRNA expressed in Met-deficient medium (GFP population 0.67, orange). Thus, the level of green emission observed in EGFP-expressing cells was approximately two-fold higher than that in methionine-free HeLa cells. These results demonstrated that E. coli Met-tRNAi can be used for protein synthesis in methionine-free cells, providing experimental proof of initial translation using a fluorescence-labeled, methionine-charged human tRNAi probe.

The expression level of EGFP mRNA increased after the introduction of EGFP-transfected HeLa cells; however, l-Met deficiency and l-Met sufficiency acted as positive controls in correlation with the HeLa cell viability (Fig. 3B). Thus, the effect of l-Met on HeLa cell viability occurs during the initial translation stage. The decrease in the viability of HeLa cells without methionine was not significant compared with that of control HeLa cells with sufficient methionine (Fig. 3B), providing experimental proof of the Hoffman effect [33], whereby malignant cells can endogenously synthesize high levels of methionine, but not to a sufficient level to sustain cancer growth.

EGFP expression via the initiation of the translational reading frame with E. coli Met-tRNAi/ EcMRS pairs

To evaluate the activity of E. coli tRNAi, EGFP was expressed by transfecting HeLa cells in an l-Met–free medium. The E. coli Met-tRNAi/EcMRS-charged E. coli tRNAi, used as a bio-orthogonal reaction, was paired with proteins produced in eukaryotic hosts (Fig. 4A). The EGFP mRNA levels determined using RT-qPCR demonstrated a correlation with the E. coli Met-tRNAi/EcMRS pairs. In the experiment involving E. coli Met-tRNAi/EcMRS pairs alone, EGFP mRNA expression (blue bars), EGFP expression dot plots (Supplementary Figure S4, GFP group 0.58, blue), and EGFP mRNA expression induced with l-Met were observed in methionine-deficient medium (Fig. 4A; GFP population 0.35, orange). In addition, EGFP mRNA expression was observed after introducing E. coli Met-tRNAi/EcMRS pairs alone with l-Met–sufficient HeLa cells used as a control (Supplementary Figure S4: GFP population 0.01, red). Figure 4B shows HeLa cell viability induced by the expression of EGFP mRNA. In HeLa cells without methionine, the cell viability introduced by adding 1, 5, and 10 nM of the E. coli Met-tRNAi/EcMRS pair (blue bar) was dependent on the concentration change of the HeLa cell control with sufficient methionine. The E. coli Met-tRNAi/EcMRS pair showed significant toxicity at a concentration of 10 nM (Fig. 4B).

These findings suggest the possibility of an association with the EGFP mRNA expression level introduced by the E. coli Met-tRNAi/EcMRS pair (blue bar). Surprisingly, the expression of EGFP mRNA decreased after introduction of the E. coli Met-tRNAi/HMRS pair (green bar), in contrast to the E. coli Met-tRNAi/EcMRS (blue bar) pair comparison experiment (Fig. 4). The expression of EGFP mRNA increased after introduction of the E. coli Met-tRNAi/HMRS pairs (green bars), as shown in Fig. 4C. The expression of E. coli Met-tRNAi/HMRS pairs was higher under methionine-deficient HeLa cell conditions than that observed when using E. coli Met-tRNAi/EcMRS pairs (Fig. 4C, GFP population 14.8, green). The expression of EGFP mRNA decreased after introduction of the E. coli MRS pair. The results for the HMRS group were compared with those of the introduction of an E. coli initiator charged with EcMRS (Fig. 4C; GFP population 4.81, blue) as a replacement for HMRS (Fig. 4C, GFP population 14.8, green), demonstrating that the expression of GFP mRNA increased after the introduction of EGFP expression.

Comparison of the effect of formylated methionine-conjugated and non-formylated methionine-conjugated E. coli RNAi on the survival of HeLa cells

To determine the viability of E. coli tRNAi in non-formylated methionine used in the initial translational reading frame, samples with non-formylated and formylated methionine were compared by monitoring the viability of HeLa cells. Human cells exhibited detectable, non-formylated methionine-charged E. coli tRNAi/MRS complex responses, and compared with endogenous human Met-tRNAi, human cells via E. coli initiator/human MRS initiated synthesis.

In methionine-free HeLa cells, non-formylated methionine-conjugated E. coli Met-tRNAi/EcMRS pairs (1, 5, and 10 nM) exhibited a significantly higher level of toxicity compared with that seen in formylated methionine-conjugated E. coli Met-tRNAi/HMRS pairs (Fig. 5 and Supplementary Figure S5). Interestingly, non-formylated methionine bound to E. coli tRNAi was associated with lower cell viability compared with that seen with formylated methionine. The non-formylated E. coli Met-tRNAi is rooted in the RNA (tRNAi)/aaRS pair complex structure and exhibits a maintenance function of the reading frame for translation initiation. Treatment of non-formylated Met-E. coli tRNAi in repressed conditions by depleting l-methionine reduces the protein expression rate induced by the initiation of protein synthesis relative to repressing HeLa cell viability. Non-formylated Met-E. coli tRNAi/EMRS pairs led to lower cell viability in HeLa cells due to weaker protein synthesis efficiency. Thus, our results suggest that the ability of E. coli tRNAi motifs to initiate protein synthesis may be a key player in cell growth.

Verification of the quality of single fluorescent methionine-conjugated E. coli initiator tRNAs

We proceeded to assess E. coli fluorescent Met-tRNAi, using the employed SEC method to separate unlabeled methionine-conjugated E. coli tRNAi and 5-FAM or methionine (Fig. 6). The peaks shown on the left of Fig. 6a represent free methionine (210 nm, 13 min), the fluorescent label alone (210 nm, 12 min), and the fluorescence detector (FLD). We previously reported the N-terminal labeling of target proteins with the strong chromophore Cy5-Met [54–57]. Purified Cy5-Met was chemically synthesized and then purified to validate the occurrence of protein synthesis mediated by single fluorescent methionine-conjugated E. coli tRNAi. We also chemically synthesized 5-FAM-Met, which was then coupled to synthetic E. coli tRNAi, as demonstrated by magnetic resonance spectroscopy. E. coli tRNAi was generated via in vitro transcription and separated (according to mass) from 5-FAM (473.39 Da), E. coli tRNAi (26.07 kDa), and methionine alone (149 Da) (Fig. 6A). Here, 5-FAM-labeled methionine-charged E. coli tRNAi clearly overlaid the fluorescence detection spectra and could be distinguished by its absorbance peak of 210 nm at 5.3 min (Fig. 6B) and a peak at 260 nm that also corresponded to 5.3 min.

The peak corresponding to 5.3 min was further analyzed via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Fig. 6C and Supplementary Figure S2). As an additional control, we compared the methionylation of fluorescent methionine-conjugated human tRNAi with fluorescent methionine-conjugated E. coli tRNAi using 2% agarose gel electrophoresis, followed by imaging with 260-nm UV irradiation and FLD (Supplementary Figure S3). To study the effect of the non-formylated methionine-conjugated E. coli tRNAi on translation recognition in HeLa cells, we purified EcMRS based on its affinity for fluorescent methionine-conjugated E. coli tRNAi. This enzyme is activated by the amine group of unpurified fluorescent methionine, meaning that non-formylated, rather than formylated, methionine is charged by EcMRS. The effect on a single fluorescent methionine-binding E. coli initiator tRNA was measured for fluorescently labeled synthetic proteins using E. coli Met-tRNAi/EcMRS.

The present study demonstrated the occurrence of protein synthesis through initiation of the translational reading frame using the E. coli tRNAi/aaRS pair. In a previous study, the POI-induced decreased GFP mRNA expression in human Met-tRNAi/HMRS pairs confirmed the expression of GFP[37]. Nevertheless, endogenous human tRNA was replaced by E. coli RNAi, confirming the expression of GFP in l-methionine-deficient HeLa cells. This study investigated the effect of non-formylated E. coli Met-tRNAi on POI synthesis in HeLa cells under depletion of intracellular L-Met. We discovered that the selective linking of fluorescent methionine to E. coli Met-tRNAi was highly precise, as shown in Fig. 6. Moreover, every EcMRS paired with Met could be promoted as a newly synthesized protein in HeLa cells.

tRNA plays a crucial role in research for understanding protein translation, protein synthesis, and the evolution from RNA to modern cellular forms during the early diversification of living organisms [58]. Although accurate measurement of the expression rate of specific newly synthesized proteins remains controversial, the discovery of a newly synthesized protein regulator may be advantageous for the treatment of human diseases. In all eukaryotic and archaeal tRNAs, eukaryotic cytoplasmic tRNAi is characterized by the anticodon sequence CUA being preceded by C but not U [12, 45]. The N-terminal formylated methionine of a new protein is deformed, and the resulting methionine can be cleaved off by methionine aminopeptidase (MetAP) in E. coli [59, 60]. The removal of deformylmethionine from MetAP requires a residue to become an Nterminal after cleavage[59, 61].

As seen in Fig. 2, despite the removal of deformylated methionine, the EGFP expression level can serve as a positive control when using E. coli tRNAi and human tRNAi. The function of endogenous human Met-tRNAi can be inhibited by the depletion of intracellular l-Met in l-Met–deficient HeLa cells, which can be attributed to two types of cells: non-formylated Met-E. coli tRNAi/EcMRS and non-formylated Met-E. coli tRNAi/HMRS. In other words, we showed that E. coli Met-tRNAi is associated with cytoplasmic protein translation in mammalian systems based on its aminoacylation and the recognition of E. coli Met-tRNAi by EcMRS. To monitor corrective protein synthesis, proteins can be subjected to a change in conditions, such as the accurate pairing of cognate tRNAs, stimuli, or various stresses, after which the synthesized protein rates can be measured.

Expansion of tRNA species facilitates correct or mistranslation of the reading frame, selective genetic code, encoding translation, and engineered E. coli Met-tRNAi to greatly improve our understanding of E. coli tRNAs. Notably, MRS is also an important enzyme in the initiation of translation, and Met-tRNAi is considered a strong biomarker candidate for the treatment of cancer [62, 63]. We investigated protein synthesis by initiating the translational reading frames of E. coli tRNAi/aaRS pairs. Comparing the results obtained for MRS substitution, we found that MRS from both humans and E. coli synthesized POIs in the cytoplasm. These results indicate that various initiator tRNAs potentially act on different tRNA sequence motif interactions. MRS has a high degree of specificity for methionine and can distinguish tRNAi molecules with similar structures [31, 64, 65]. However, this process can introduce errors in protein synthesis while attaching certain amino acids to non-cognate tRNAs with a high level of accuracy [26]. Therefore, the tRNA sequence motif interaction between synthetase and tRNA is a potential factor that ensures the accuracy of the translational reading frame. These results suggest that the introduction of E. coli Met-tRNAi/EcMRS pairs in the initial translation frame for GFP mRNA expression inhibits protein initiation due to cytotoxicity.

In the absence of the introduction of protein initiation, the HeLa cell viability results suggest that Met is a complex metabolic pathway associated with cancer at multiple levels. However, the lower HeLa cell viability with non-formylated Met introduced during the initiation of protein synthesis suggests that the cytotoxicity induced in the early translation frame of GFP mRNA expression is significantly associated with EcMRS. In general, most cancer cells readily synthesize Met from homocysteine but cannot proliferate in a medium in which Met has been replaced with homocysteine. Therefore, the results depicted in Fig. 5 suggest that the increased viability of HeLa cells cultured under Met-deficient conditions could mean that supplementation with homocysteine [66] is associated with increased HeLa cell viability. Human tumor cells depend on exogenous methionine, whereas normal tissues may depend on homocysteine to meet methionine needs [66, 67]. This suggests that malignant cells can adapt to glucose-poor conditions by adopting alternative metabolic pathways that support growth [68–70]. Therefore, by introducing our methionine-deficient medium condition, the viability characteristics of methionine-dependent HeLa cells were efficiently applied to the experimental system to illustrate the significance of E. coli tRNAi-mediated initiation protein synthesis and confirm previous reports [66, 67]. These findings highlight potential codons for protein turnover by optimizing rare codons to promote protein fidelity. Moreover, this approach can be used to accurately track many types of aaRS through pairing with cognate tRNAs to investigate the fidelity of newly synthesized protein translation.

In conclusion, our results suggest that the ability of E. coli tRNAi motifs to initiate protein synthesis may be a potential key player in cell growth. Therefore, these results provide new insights into the optimization of tRNAi motifs to allow control over the initiation of protein synthesis. Moreover, our observations of the fluorophore-E. coli Met-tRNAi bases further advance the understanding of protein expression in eukaryotic cells by revealing the various tRNAi sequence motifs involved in protein synthesis and fidelity. Accordingly, the results of this study could be used to develop a strategy for the optimization of tRNAi motifs to facilitate precise protein synthesis. In particular, this work suggests a novel potential approach for the early suppression of cells before the cell migration associated with increased cell growth rates. This approach differs from traditional treatment strategies and is suggested as a scheme for controlling the initiation of protein synthesis relative to cell viability. Therefore, this study provides new insights on non-formylated Met-tRNAi with the potential to control the synthesis of various biomarker proteins in eukaryotic cells.

Conflict of interest

All contributing authors declare that they have no conflicts of interest.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Statistical analysis

All values are expressed as means. Error bars indicate the standard deviation. Graph Adobe CS7 software was used for graphing and statistical analysis.

Data and materials availability

All data discussed in this paper are available in the main text and Supplementary Information. The materials used in this study are available upon request from the corresponding authors.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF 2019R1I1A1A01043047).

Author Contributions: J.M. Kim designed the research and conducted the experiments. J.M. Kim wrote the paper. J.M. Kim and Y-M Kang conducted the experiments.

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Protein of Interest Synthesis Using Non-formylated Methionine−Escherichia coli initiator tRNA in HeLa cells

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Abstract

Figures

Introduction

Materials And Methods

Materials

In vitro human initiator tRNA and E. coli initiator tRNAs synthesis

Modified initiator tRNA methionylation

Isolation of fluorescent-labeled methionine-charged initiator tRNA

Analysis of L-methionine (L-Met) and fluorophore-conjugated methionine with high-performance liquid chromatography

Cell culture and enhanced green fluorescent protein (EGFP) transfection via methionine presence in media

Construction of expression plasmid (EGFP)

Flow cytometry for determination of E. coli initiator tRNA activity

qRT-PCR

Mass analysis of the modified initiator tRNA

Fluorescent and biotin labeling Tat protein detection

Cell viability assay

Statistical analysis

Results

Discussion

Declarations

References

Supplementary Files

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