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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 mutants display reduced wing size.(a) Adult wing phenotypes induced by PheRS-sd mutations. en-Gal4 (en) was used to drive transgene expression in the posterior compartment of developing wing discs. The dark dashed line indicates the border of the anterior/posterior compartment. In the PheRS wild-type background, wings expressing GFP and βA158W were normal. The ones expressing αA456G showed vein defects (arrowhead) and also reduced wing size in the posterior compartment. In the gβPheRS-rescued β-PheRS background, wings expressing GFP and αA456G, respectively, showed similar phenotypes as in the PheRS+ background. In contrast, wings expressing αA456G in a gβA158W background displayed severe size reduction with significant parts of the organ missing altogether. Scale bars represent 500 μm. (b,c) Quantification of wing sizes from the experiment shown in a. Twelve single female wings from different flies were analysed for each genotype. Wing size was measured by counting the pixels in an anterior (A; outlined in blue) and a posterior area (P; outlined in yellow) of each wing, and the ratio (P/A) was calculated. (b) In a PheRS+ wild-type background, αA456G mutants showed a size decrease, but βA158W did not. (c) In the rescued β-PheRS background, the αA456G mutant showed a size decrease and, when αA456G and βA158W are expressed, wing size was further decreased. Error bars represent s.e.m. n=12, n.s., not significant; ***P<0.001, t-test.
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f5: PheRS-sd mutants display reduced wing size.(a) Adult wing phenotypes induced by PheRS-sd mutations. en-Gal4 (en) was used to drive transgene expression in the posterior compartment of developing wing discs. The dark dashed line indicates the border of the anterior/posterior compartment. In the PheRS wild-type background, wings expressing GFP and βA158W were normal. The ones expressing αA456G showed vein defects (arrowhead) and also reduced wing size in the posterior compartment. In the gβPheRS-rescued β-PheRS background, wings expressing GFP and αA456G, respectively, showed similar phenotypes as in the PheRS+ background. In contrast, wings expressing αA456G in a gβA158W background displayed severe size reduction with significant parts of the organ missing altogether. Scale bars represent 500 μm. (b,c) Quantification of wing sizes from the experiment shown in a. Twelve single female wings from different flies were analysed for each genotype. Wing size was measured by counting the pixels in an anterior (A; outlined in blue) and a posterior area (P; outlined in yellow) of each wing, and the ratio (P/A) was calculated. (b) In a PheRS+ wild-type background, αA456G mutants showed a size decrease, but βA158W did not. (c) In the rescued β-PheRS background, the αA456G mutant showed a size decrease and, when αA456G and βA158W are expressed, wing size was further decreased. Error bars represent s.e.m. n=12, n.s., not significant; ***P<0.001, t-test.

Mentions: Translation is a major force for cellular growth. We therefore also assessed the effect of the PheRS-sd mutations on growth. The Drosophila wing is a proven system to study growth control in vivo. We used the engrailed-Gal4 (en-Gal4) driver to express our test genes in the posterior compartment of the wing disc. This allowed us to compare the effect on the posterior cells to the wild-type control cells in the anterior compartment of the very same organ. As seen in Fig. 5a, expressing the αA456G mutation caused vein defects, while expression of the βA158W mutation did not cause any detectable wing phenotypes. However, expressing both, the mutant αA456G and the mutant βA158W, simultaneously in the posterior compartment caused a dramatic decrease in wing size, and in some wings entire regions of the posterior compartment were missing. This feature is reminiscent of the enhanced effect caused by expression of both mutations during eye development.


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 mutants display reduced wing size.(a) Adult wing phenotypes induced by PheRS-sd mutations. en-Gal4 (en) was used to drive transgene expression in the posterior compartment of developing wing discs. The dark dashed line indicates the border of the anterior/posterior compartment. In the PheRS wild-type background, wings expressing GFP and βA158W were normal. The ones expressing αA456G showed vein defects (arrowhead) and also reduced wing size in the posterior compartment. In the gβPheRS-rescued β-PheRS background, wings expressing GFP and αA456G, respectively, showed similar phenotypes as in the PheRS+ background. In contrast, wings expressing αA456G in a gβA158W background displayed severe size reduction with significant parts of the organ missing altogether. Scale bars represent 500 μm. (b,c) Quantification of wing sizes from the experiment shown in a. Twelve single female wings from different flies were analysed for each genotype. Wing size was measured by counting the pixels in an anterior (A; outlined in blue) and a posterior area (P; outlined in yellow) of each wing, and the ratio (P/A) was calculated. (b) In a PheRS+ wild-type background, αA456G mutants showed a size decrease, but βA158W did not. (c) In the rescued β-PheRS background, the αA456G mutant showed a size decrease and, when αA456G and βA158W are expressed, wing size was further decreased. Error bars represent s.e.m. n=12, n.s., not significant; ***P<0.001, t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: PheRS-sd mutants display reduced wing size.(a) Adult wing phenotypes induced by PheRS-sd mutations. en-Gal4 (en) was used to drive transgene expression in the posterior compartment of developing wing discs. The dark dashed line indicates the border of the anterior/posterior compartment. In the PheRS wild-type background, wings expressing GFP and βA158W were normal. The ones expressing αA456G showed vein defects (arrowhead) and also reduced wing size in the posterior compartment. In the gβPheRS-rescued β-PheRS background, wings expressing GFP and αA456G, respectively, showed similar phenotypes as in the PheRS+ background. In contrast, wings expressing αA456G in a gβA158W background displayed severe size reduction with significant parts of the organ missing altogether. Scale bars represent 500 μm. (b,c) Quantification of wing sizes from the experiment shown in a. Twelve single female wings from different flies were analysed for each genotype. Wing size was measured by counting the pixels in an anterior (A; outlined in blue) and a posterior area (P; outlined in yellow) of each wing, and the ratio (P/A) was calculated. (b) In a PheRS+ wild-type background, αA456G mutants showed a size decrease, but βA158W did not. (c) In the rescued β-PheRS background, the αA456G mutant showed a size decrease and, when αA456G and βA158W are expressed, wing size was further decreased. Error bars represent s.e.m. n=12, n.s., not significant; ***P<0.001, t-test.
Mentions: Translation is a major force for cellular growth. We therefore also assessed the effect of the PheRS-sd mutations on growth. The Drosophila wing is a proven system to study growth control in vivo. We used the engrailed-Gal4 (en-Gal4) driver to express our test genes in the posterior compartment of the wing disc. This allowed us to compare the effect on the posterior cells to the wild-type control cells in the anterior compartment of the very same organ. As seen in Fig. 5a, expressing the αA456G mutation caused vein defects, while expression of the βA158W mutation did not cause any detectable wing phenotypes. However, expressing both, the mutant αA456G and the mutant βA158W, simultaneously in the posterior compartment caused a dramatic decrease in wing size, and in some wings entire regions of the posterior compartment were missing. This feature is reminiscent of the enhanced effect caused by expression of both mutations during eye development.

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