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Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development.

Lu J, Bergert M, Walther A, Suter B - Nat Commun (2014)

Bottom Line: At the cellular level, the mutations reduce cell proliferation and promote cell death.Our results also reveal the particular importance of the first amino-acid recognition sieve.Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Bern 3012, Switzerland.

ABSTRACT
Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

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PheRS-sd mutations induce apoptosis and reduce cell proliferation.(a) Confocal microscopy images of wing discs from third instar wandering larvae. The posterior compartment is marked with GFP in green. CP3 is visualized in red with anti-CP3 antibodies. The anterior wing disc compartment (GFP-negative) serves as internal control. All flies shown are in the β-PheRS background, rescued by gβPheRS or gβA158W. A few CP3-positive cells were observed in αA456G mutant wing discs; however, many more cells underwent apoptosis when αA456G and βA158W mutants were expressed. Some CP3-positive cells are pointed out by arrowheads. Note that CP3-positive cells (red) always overlap with GFP (green). DNA is shown in blue. Scale bars represent 50 μm. (b) Quantification of apoptotic wing discs. No apoptosis was detectable in wing discs from controls and wing discs expressing only βA158W. A small number of wing discs expressing αA456G showed apoptotic cells. Apoptosis was most prominent in wing discs that expressed αA456G and βA158W mutant protein. (c,d) Wing discs were subjected to FACS analysis. The GFP signal was used to distinguish cell populations that express the transgenes. (c) The αA456G mutation reduces cell numbers. Cell numbers were determined relative to the cell numbers in the anterior (GFP-negative) area. Error bars indicate s.e.m. n=3, **P<0.01, t-test. (d) The αA456G mutation does not reduce cell size. Cell size was determined by Forward Scatter. Cells from the GFP-positive compartment are smaller than the ones from the GFP-negative compartment in the control. In αA456G mutants, their sizes are similar. Figure shows representatives of three independent experiments.
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f6: PheRS-sd mutations induce apoptosis and reduce cell proliferation.(a) Confocal microscopy images of wing discs from third instar wandering larvae. The posterior compartment is marked with GFP in green. CP3 is visualized in red with anti-CP3 antibodies. The anterior wing disc compartment (GFP-negative) serves as internal control. All flies shown are in the β-PheRS background, rescued by gβPheRS or gβA158W. A few CP3-positive cells were observed in αA456G mutant wing discs; however, many more cells underwent apoptosis when αA456G and βA158W mutants were expressed. Some CP3-positive cells are pointed out by arrowheads. Note that CP3-positive cells (red) always overlap with GFP (green). DNA is shown in blue. Scale bars represent 50 μm. (b) Quantification of apoptotic wing discs. No apoptosis was detectable in wing discs from controls and wing discs expressing only βA158W. A small number of wing discs expressing αA456G showed apoptotic cells. Apoptosis was most prominent in wing discs that expressed αA456G and βA158W mutant protein. (c,d) Wing discs were subjected to FACS analysis. The GFP signal was used to distinguish cell populations that express the transgenes. (c) The αA456G mutation reduces cell numbers. Cell numbers were determined relative to the cell numbers in the anterior (GFP-negative) area. Error bars indicate s.e.m. n=3, **P<0.01, t-test. (d) The αA456G mutation does not reduce cell size. Cell size was determined by Forward Scatter. Cells from the GFP-positive compartment are smaller than the ones from the GFP-negative compartment in the control. In αA456G mutants, their sizes are similar. Figure shows representatives of three independent experiments.

Mentions: We next tested whether PheRS-sd mutations cause reduced wing size by inducing cell death. Staining wing discs for the CP3 epitope, we identified a small number of CP3-positive cells in cases where we expressed the αA456G mutation (Fig. 6a). Expressing the αA456G and the βA158W mutant simultaneously, we observed a much higher percentage of CP3-positive cells and some of these cells formed clusters rather than being dispersed over the disc. Importantly, these apoptotic cells were restricted to the posterior compartment where the PheRS-sd mutations were expressed. Confirming theses results with the TdT-mediated dUTP nick end labelling (TUNEL) assay (Supplementary Fig. 1), we conclude that apoptosis contributed to the impaired organ size.


Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development.

Lu J, Bergert M, Walther A, Suter B - Nat Commun (2014)

PheRS-sd mutations induce apoptosis and reduce cell proliferation.(a) Confocal microscopy images of wing discs from third instar wandering larvae. The posterior compartment is marked with GFP in green. CP3 is visualized in red with anti-CP3 antibodies. The anterior wing disc compartment (GFP-negative) serves as internal control. All flies shown are in the β-PheRS background, rescued by gβPheRS or gβA158W. A few CP3-positive cells were observed in αA456G mutant wing discs; however, many more cells underwent apoptosis when αA456G and βA158W mutants were expressed. Some CP3-positive cells are pointed out by arrowheads. Note that CP3-positive cells (red) always overlap with GFP (green). DNA is shown in blue. Scale bars represent 50 μm. (b) Quantification of apoptotic wing discs. No apoptosis was detectable in wing discs from controls and wing discs expressing only βA158W. A small number of wing discs expressing αA456G showed apoptotic cells. Apoptosis was most prominent in wing discs that expressed αA456G and βA158W mutant protein. (c,d) Wing discs were subjected to FACS analysis. The GFP signal was used to distinguish cell populations that express the transgenes. (c) The αA456G mutation reduces cell numbers. Cell numbers were determined relative to the cell numbers in the anterior (GFP-negative) area. Error bars indicate s.e.m. n=3, **P<0.01, t-test. (d) The αA456G mutation does not reduce cell size. Cell size was determined by Forward Scatter. Cells from the GFP-positive compartment are smaller than the ones from the GFP-negative compartment in the control. In αA456G mutants, their sizes are similar. Figure shows representatives of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263187&req=5

f6: PheRS-sd mutations induce apoptosis and reduce cell proliferation.(a) Confocal microscopy images of wing discs from third instar wandering larvae. The posterior compartment is marked with GFP in green. CP3 is visualized in red with anti-CP3 antibodies. The anterior wing disc compartment (GFP-negative) serves as internal control. All flies shown are in the β-PheRS background, rescued by gβPheRS or gβA158W. A few CP3-positive cells were observed in αA456G mutant wing discs; however, many more cells underwent apoptosis when αA456G and βA158W mutants were expressed. Some CP3-positive cells are pointed out by arrowheads. Note that CP3-positive cells (red) always overlap with GFP (green). DNA is shown in blue. Scale bars represent 50 μm. (b) Quantification of apoptotic wing discs. No apoptosis was detectable in wing discs from controls and wing discs expressing only βA158W. A small number of wing discs expressing αA456G showed apoptotic cells. Apoptosis was most prominent in wing discs that expressed αA456G and βA158W mutant protein. (c,d) Wing discs were subjected to FACS analysis. The GFP signal was used to distinguish cell populations that express the transgenes. (c) The αA456G mutation reduces cell numbers. Cell numbers were determined relative to the cell numbers in the anterior (GFP-negative) area. Error bars indicate s.e.m. n=3, **P<0.01, t-test. (d) The αA456G mutation does not reduce cell size. Cell size was determined by Forward Scatter. Cells from the GFP-positive compartment are smaller than the ones from the GFP-negative compartment in the control. In αA456G mutants, their sizes are similar. Figure shows representatives of three independent experiments.
Mentions: We next tested whether PheRS-sd mutations cause reduced wing size by inducing cell death. Staining wing discs for the CP3 epitope, we identified a small number of CP3-positive cells in cases where we expressed the αA456G mutation (Fig. 6a). Expressing the αA456G and the βA158W mutant simultaneously, we observed a much higher percentage of CP3-positive cells and some of these cells formed clusters rather than being dispersed over the disc. Importantly, these apoptotic cells were restricted to the posterior compartment where the PheRS-sd mutations were expressed. Confirming theses results with the TdT-mediated dUTP nick end labelling (TUNEL) assay (Supplementary Fig. 1), we conclude that apoptosis contributed to the impaired organ size.

Bottom Line: At the cellular level, the mutations reduce cell proliferation and promote cell death.Our results also reveal the particular importance of the first amino-acid recognition sieve.Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Bern 3012, Switzerland.

ABSTRACT
Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

Show MeSH