Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex

Abstract

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.

Figure 1.

The leucine zipper of genetically encoded arginyl-tRNA synthetase can be skipped by genome editing. (A) Full-length ArgRS expression was disrupted by introducing a frameshift in exon 2. ArgRS can subsequently be only expressed as a truncated form lacking the first 1–72 amino acids through initiation from a second ATG start codon (red arrow) as shown by western blot. Black arrow: Full-length ArgRS. A representative out of 5 repeats is shown. Scheme of the ArgRS protein. ArgRS leucine zipper: blue, ArgRS N-terminal, catalytic, and tRNA binding domain: red. (B) Peptide coverage following mass spectrometric detection of ArgRS enriched from wild type or gRNA_RARS1_ex2 cells confirming the loss of the ArgRS leucine zipper (ΔLZ). Peptides found only in wildtype cells expressing mostly full-length ArgRS are shown in blue, while peptides detected in both or only in ΔLZ cells are red. Black peptides were not detected. Bold: alternative start codon. Coverage was mapped from three repeats.

Figure 2.

Deletion of the ArgRS LZ excludes ArgRS and GlnRS from the MSC. (A-C) Volcano plot of proteins identified by mass spectrometry following co-immunoprecipitation of MetRS interaction partners (3 repeats). The number of identified unique and razor peptides is visualized by color. Interaction partners of methionyl-tRNA synthetase (MetRS), a protein in the multisynthetase complex, in HEK 293T (A) wildtype and (B) ΔLZ cells. (C) Wildtype vs ΔLZ cells: ArgRS and GlnRS are lost from the MetRS interactome upon loss of the ArgRS LZ.

Figure 3.

The MSC persists as a macromolecular complex in the absence of ArgRS and GlnRS. Cell lysate was separated by size exclusion chromatography to distinguish between monomeric/dimeric and multisynthetase complex-bound proteins. aaRSs were identified by western blot. Intact multisynthetase complex elutes between fraction 10–12. Dimeric or monomeric tRNA synthetases elude between fraction 14–17. (A–D) wt: wildtype 293T cells, ΔLZ: 293T cells expressing only the truncated version of ArgRS lacking the N-terminal leucine zipper. (A) Arginyl-tRNA synthetase, ArgRS. Exposure times differ to accommodate for lower expression levels of ArgRS ΔLZ. (B) Glutaminyl-tRNA synthetase, GlnRS. (C) Methionyl-tRNA synthetase, MetRS. (D) Tyrosyl-tRNA synthetase, TyrRS. (E–G) The leucine zipper of ArgRS was fused to the N-terminus of mCerulean (LZ-mCer). Monomeric mCerulean elutes between fraction 17–18. mCer: mCerulean, cyan fluorescent protein. ΔLZ_mCer: mCerulean with the leucine zipper of ArgRS fused to its N-terminus. Scheme of the LZ-mCerulean fusion protein and ArgRS variant. ArgRS LZ: blue, ArgRS N-terminal, catalytic and tRNA binding domain: red. mCerulean: light blue. (E) mCerulean (detected by an anti-GFP antibody). (F) Glutaminyl-tRNA synthetase, GlnRS. (G) Methionyl-tRNA synthetase, MetRS.

Figure 4.

Loss of ArgRS and GlnRS from the multisynthetase complex does not affect tRNA charging, mRNA translation, or cell viability. (A–E) wt: wildtype 293T cells, ΔLZ: 293T cells expressing only the truncated version of ArgRS lacking amino acid 1–72. LZ-mCer: the ArgRS leucine zipper was fused to mCerulean and rescues the loss of GlnRS from the MSC. (A) Charged versus uncharged Arg- and Gln-tRNA levels by northern blot (three repeats). The aminoacylation state of the tRNA can be preserved by acidic conditions (pH 5), while the aminoacyl-tRNA-bond hydrolyzes at neutral or basic pH (pH 9). (B) Cell viability on different days after seeding was assessed by Alamar Blue. Mean of quadruplets are shown with standard derivation. A representative out of three repeats is shown. (C) Alamar blue cell viability assay in different arginine concentrations after 24 h. Experiments were performed in technical quadruplets and a representative out of three repeats is shown. (D) Western blot detection of puromycin incorporation to visualize newly synthesized proteins. Arginine-fold changes are relative to DMEM arginine levels. A representative out of four repeats is shown. (E) Western blot detection of puromycin incorporation under cell stress. CHX: cycloheximide, ribosome inhibitor. MG132: proteasome inhibitor. Torin: mTORC1 inhibitor. 17-AAG: Hsp90 inhibitor. A representative out of four repeats is shown.

Figure 5.

No indication for ribosome stalling on cognate codons upon loss of ArgRS and/or GlnRS from the MSC. (A–D) Analysis of ribosome footprints for pausing on arginine or glutamine codons. wt: wildtype 293T cells, ΔLZ: 293T cells expressing only the truncated version of ArgRS lacking the N-terminal leucine zipper (LZ), LZ_mCer: ArgRS LZ fused to mCerulean. Three repeats. (A, B) Observed ribosome occupancy versus expected occupancy based on genetic codon usage in the E, P, and A-site of the ribosome in arginine (A) and glutamine (B) codons calculated from ribosome footprints. (C, D) Percentage of arginine (C) and glutamine (D) codons among all codons with a pause score P ≥ 10 calculated from ribosome footprints.

Figure 6.

Loss of ArgRS leucine zipper lowers nuclear levels of MSC aaRSs. (A, B) Arginyl-tRNA synthetase, ArgRS. Glutaminyl-tRNA synthetase, GlnRS. Lysyl-tRNA synthetase, LysRS. Methionyl-tRNA synthetase, MetRS. Glycyl-tRNA synthetase, GlyRS. Tyrosyl-tRNA synthetase, TyrRS. GAPDH: cytoplasm loading control. Histone H3: nuclear loading control. Western blots were imaged with different exposures for cytoplasmic and nuclear fractions. (A) Western blot of cytoplasmic and nuclear fraction from wildtype cells and cells with truncated ArgRS (ΔLZ). (B) Western blot of the cytoplasmic and nuclear fraction from wildtype cells, cells with truncated ArgRS (ΔLZ), and ΔLZ cells expressing ΔLZ_mCer.