Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase

Abstract

The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5'-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases.

Keywords: IRES; aminoacyl-tRNA synthetase; post-transcriptional control; uORF.

Figure 1.

Organization of human GARS mRNAs. (A). Schematic representation of the 2 isoforms of the human GARS mRNA revealed in this study. The sequence identified by Mudge and collaborators in 1998 in fetal liver cells was used as a reference. The long mRNA1 contains 3 initiation codons, AUG₀ (initiates the synthesis of the uORF), AUG_mito (initiates translation of the mitochondrial targeting signal -MTS- fused to GARS), AUG_cyto (initiates translation of the cytosolic GARS) and a UAG stop codon (ends translation of the uORF). The shorter mRNA2 contains only AUG_mito and AUG_cyto. (B). Multiple alignment of mammalian mRNA1 5′-UTR: blasting the nucleotide sequence of Homo sapiens GARS mRNA1 (NC_018918.2) against all NCBI nucleotide databases (http://blast.ncbi.nlm.nih.gov/Blast) retrieved 43 sequences only from other mammalian genomes. Among them, 14 GARS sequences were from primates. Here, is shown only a representative selection of the retrieved sequences: Homo-sapiens (NC_018918.2), Gorilla-gorilla (NC_018431.1), Pan-troglodyte (NC_006474.3), Nomascus-leucogenys (NC_019832.1), Pongo-abelii (NC_012598.1), Macaca-mulatta (NC_007860.1), Cavia-porcellus (XM_003467957.2), Pteropus-alecto (XM_006912040.1), Mustela-putorius-furo (XM_004762573.1), Ceratotherium-simum (XM_004418866.1), Ovis-aries (XM_004007927.1), Bos-Taurus (NM_001097566.1), Orycteropus-afer (XM_007945456.1), Odobenus-rosmarus-divergens (XM_004413949.1), Orcinus-orca (XM_004269943.1), Mus-musculus (NC_000072.6), Rattus-norvegicus (NC_005103.3) GARS sequences. Sequence alignments were computed with Tcoffee software (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi). The 3 AUG codons, AUG₀, AUG_mito and AUG_cyto, are boxed in yellow, the stop codon in red. Absence of these specific codons is indicated in gray on the alignment. The schematic representation of the 5′- end of mRNA1, used in this manuscript, is indicated: AUGs are signaled with circles, the uORF stop codon with a red cross, and the frameshift deletion with a green bar.

Figure 2.

Translation of both mRNA isoforms in vivo and in vitro. (A). Top panel shows Western blot analysis of COS-7 cells transfected with pcDNA3.1 encoding mRNA1 or mRNA2 fused to a V5 epitope nucleotide sequence on their 3′-ends. Translated GARS-V5 proteins are detected by anti-V5 antibodies. Detection of the 42 kDa ß-actin (anti-actin antibodies) was included as a loading control. Bottom panel corresponds to translation of radioactive GARSs from mRNA1 and mRNA2 in vitro, accomplished using wheat germ extracts. (B). GARS-V5 was detected by immunofluorescence, using anti-V5 antibodies coupled to FITC (green). Mitochondria were stained with Mitotracker Orange CMTMRos (red) and nuclei were stained with DAPI (blue). The final localization of GARS is specified for each mRNA. For the mRNA1 scheme, please refer to the end of the Figure 1 legend.

Figure 3.

Immunolocalization of GARS expressed from mutated mRNA1. (A). Six mutants (a to h) were generated in mRNA1 where the different AUG codons were tested for translation. GARS-V5, mitochondria and nuclei detection were performed as indicated in the legend of Figure 2 and the mRNA1 scheme is described at the end of the Figure 1 legend. The final localization of GARS is specified for each mRNA. Corresponding co-localization statistics are indicated in Figure S3A. (B). GARS expression levels (Western blot). Mutant g shows 2 bands (extra-long GARS and cytosolic GARS), indicating that the protein is only partially matured inside the mitochondria. In mutants a, b, d and h, all missing AUG_cyto, initiation of a shorter GARS is observed, which is not detectable in immunolocalization experiments. This short form of the protein is observed only in vitro and appears when initiation is very inefficient. It shows (i) that the mRNA is present in the test and (ii) that the ribosome scans until it finds the next initiator codon. In the specific case of mutant h, its expression is so low that 10 times more protein extract was loaded in hx10 to detect it. Detection of the 42 kDa ß-actin (anti-actin antibodies) was included as a loading control. (C). The same mutants were used in in vitro translation experiments (rabbit reticulocyte extracts) in the presence of ³⁵S methionine. Mutant h was not tested in vitro.

Figure 4.

Cap-independent versus cap-dependent initiation. (A). In vitro translation reactions of mRNA1 and mRNA2 were performed with wheat germ extracts. Both mRNAs were capped either with the natural m7G or the inhibitor cap analog Ap3G. Synthesized GARSs correspond to the mitochondrial (upper band) and cytosolic (lower band) enzymes. Expression levels of cytosolic (dark bars) and mitochondrial (light bars) GARSs were quantified relative to the cytosolic GARS translated from mRNA2. Error bars were calculated from 3 independent experiments. (B). Western blot analysis: Effect of a strong hairpin structure on GARS expression: COS-7 cells were transfected with pCDNA3.1 constructs containing a strong hairpin structure introduced at the 5′-end of mRNA1 and mRNA2 (HP mRNA1 and HP mRNA2, respectively).

Figure 5.

Comparison of mRNA1 and Vcip IRES elements for their ability to initiate translation. (A). Schematic representation of biscistronic pRF constructs: sequences corresponding to the Vcip IRES and mRNA1 5′-end (containing the 3 AUG codons) were inserted in the intercistronic region between the renilla and firefly ORFs (pR_VcipF and pR_mRNA1F). The cap structure at the 5′-end of the bicistronic mRNA is indicated with a black circle. (B). pRF, pR_VcipF and pR_mRNA1F were transfected in COS-7 cells and renilla (cap dependent initiation) and firefly (IRES-mediated initiation) luciferase activities were measured. pR_VcipF and pR_mRNA1F luciferase activities were represented relative to pRF activity. (C). pRF, pR_mRNA1F, pR_mouseF and pR_VcipF were transfected in COS-7 cells and IRES-mediated initiation were measured. pR_mouseF express the mouse sequence corresponding to mRNA1 (see Fig. 1B). (D). Shorter versions of the mRNA1 IRES sequence were cloned in pRF and their respective luciferase activities were measured. Graphic representations of pR_mRNA1F, pR _mRNA1F deletants and pR_mRNA2F activities are relative to the pRF negative control. Standard deviations were calculated from 3 independent experiments. For the mRNA1 scheme, please refer to the end of the Figure 1 legend.

Figure 6.

Differential colocalization of GARS with subcellular structures, depending on mRNA isoforms. GARS-V5 was expressed from mRNA1 (left) or mRNA2 (right). GARS-V5 localizations (green) were compared with red markers for mitochondria (a and b, Mitotracker), ER protein (c and d, SRPs-DsRed) and endogenous ribosomal protein S6 (e and f, rmS6). In order to remove the background noise due to the cytosolic GARS, transfected COS-7 cells were first permeabilized with digitonin. This treatment strips the plasma membrane, so that cells lose most of their cytosolic content but retain intact mitochondria and ER. Nuclei were stained with DAPI (blue). Corresponding co-localization statistics are indicated in Figure S3B.

Figure 7.

Suggested models. Summary of GARS expression from mRNA1 and mRNA2 isoforms: mRNA2 codes both the mitochondrial (red MTS) and the cytosolic GARSs using a leaky scanning mechanism. Only 2 initiation codons are recognized in mRNA1, due to a uORF containing an IRES, which hinders mitochondrial GARS synthesis. Translation initiation at AUG₀ leads (i) to the synthesis of a short peptide (green) and (ii) to the subsequent reinitiation at AUG_cyto, allowing translation of GARS on ER-bound ribosomes (blue) and its subsequent diffusion in the cytosol.