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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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Sieving-defective mutations induce impaired locomotive performance and advanced ageing.(a,b) Negative geotaxis climbing assays were performed to study locomotive behaviour. For each genotype, three groups of male flies were tested at the indicated age. The assay was recorded as video, and the movies were analysed to determine the climbing index. (a) αA456G mutant flies displayed progressively decreasing climbing ability. Expression of the α-subunit (wild-type and αA456G mutant) was driven by Act-Gal4. (b) βA158W did not differ from the wild type during the first 5 weeks. Transgenes were in the β-PheRS background, rescued by gβPheRS or gβA158W under their endogenous promoter. Error bars show s.e.m. n=3, *P<0.05, **P<0.01, t-test. (c,d) Lifespan analysis of the male flies with the same genotype as in a,b. (c) αA456G mutant flies aged faster than wild type, and the differences became apparent already at early stages. P<0.001, log-rank test. (d) At younger age, gβA158W mutant flies aged similarly as the wild type, but at older age (more than 50 days old) the mutants aged faster. P<0.001, log-rank test. (e) Confocal microscopy images of the ventrolateral protocerebrum region of the adult fly brain. Embryos and larvae were kept at 18 °C to keep Gal80ts active to repress Gal4. With this protocol, normal adults eclosed (while the ones expressing αA456G and βA158W were lethal without Gal80ts) and these flies were then incubated at 29 °C for ~7 days, inactivating Gla80ts and activating Gal4. Elav (green) marks neuronal cells, and anti-CP3 stains apoptotic cells (red). A few CP3-positive cells were detected in neurons expressing the αA456G mutant, and many more apoptotic neurons (arrowheads) were observed when both αA456G and βA158W variants were expressed. DNA is in blue. Scale bars represent 5 μm.
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f4: Sieving-defective mutations induce impaired locomotive performance and advanced ageing.(a,b) Negative geotaxis climbing assays were performed to study locomotive behaviour. For each genotype, three groups of male flies were tested at the indicated age. The assay was recorded as video, and the movies were analysed to determine the climbing index. (a) αA456G mutant flies displayed progressively decreasing climbing ability. Expression of the α-subunit (wild-type and αA456G mutant) was driven by Act-Gal4. (b) βA158W did not differ from the wild type during the first 5 weeks. Transgenes were in the β-PheRS background, rescued by gβPheRS or gβA158W under their endogenous promoter. Error bars show s.e.m. n=3, *P<0.05, **P<0.01, t-test. (c,d) Lifespan analysis of the male flies with the same genotype as in a,b. (c) αA456G mutant flies aged faster than wild type, and the differences became apparent already at early stages. P<0.001, log-rank test. (d) At younger age, gβA158W mutant flies aged similarly as the wild type, but at older age (more than 50 days old) the mutants aged faster. P<0.001, log-rank test. (e) Confocal microscopy images of the ventrolateral protocerebrum region of the adult fly brain. Embryos and larvae were kept at 18 °C to keep Gal80ts active to repress Gal4. With this protocol, normal adults eclosed (while the ones expressing αA456G and βA158W were lethal without Gal80ts) and these flies were then incubated at 29 °C for ~7 days, inactivating Gla80ts and activating Gal4. Elav (green) marks neuronal cells, and anti-CP3 stains apoptotic cells (red). A few CP3-positive cells were detected in neurons expressing the αA456G mutant, and many more apoptotic neurons (arrowheads) were observed when both αA456G and βA158W variants were expressed. DNA is in blue. Scale bars represent 5 μm.

Mentions: To further study the long-term effect of PheRS-sd mutations, we analysed their locomotive performance by a negative geotaxis climbing assay. Ubiquitous expression of the wild-type or mutant α-subunit was driven by Act-Gal4, and the climbing capability was recorded once a week (Fig. 4a, Supplementary Movie 1). While αA456G flies showed a similar climbing ability as wild-type animals during the first week, they displayed clearly impaired locomotive performance during the subsequent weeks, and during the 4th week their climbing index decreased to about one quarter of the index of same age control flies. In contrast, the climbing ability of βA158W mutants did not differ significantly from wild-type controls (Fig. 4b). The Act-Gal4-driven expression of the αA456G in the gβA158W transgenic background turned out to be lethal during development, preventing us from examining the locomotive performance of adults.


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)

Sieving-defective mutations induce impaired locomotive performance and advanced ageing.(a,b) Negative geotaxis climbing assays were performed to study locomotive behaviour. For each genotype, three groups of male flies were tested at the indicated age. The assay was recorded as video, and the movies were analysed to determine the climbing index. (a) αA456G mutant flies displayed progressively decreasing climbing ability. Expression of the α-subunit (wild-type and αA456G mutant) was driven by Act-Gal4. (b) βA158W did not differ from the wild type during the first 5 weeks. Transgenes were in the β-PheRS background, rescued by gβPheRS or gβA158W under their endogenous promoter. Error bars show s.e.m. n=3, *P<0.05, **P<0.01, t-test. (c,d) Lifespan analysis of the male flies with the same genotype as in a,b. (c) αA456G mutant flies aged faster than wild type, and the differences became apparent already at early stages. P<0.001, log-rank test. (d) At younger age, gβA158W mutant flies aged similarly as the wild type, but at older age (more than 50 days old) the mutants aged faster. P<0.001, log-rank test. (e) Confocal microscopy images of the ventrolateral protocerebrum region of the adult fly brain. Embryos and larvae were kept at 18 °C to keep Gal80ts active to repress Gal4. With this protocol, normal adults eclosed (while the ones expressing αA456G and βA158W were lethal without Gal80ts) and these flies were then incubated at 29 °C for ~7 days, inactivating Gla80ts and activating Gal4. Elav (green) marks neuronal cells, and anti-CP3 stains apoptotic cells (red). A few CP3-positive cells were detected in neurons expressing the αA456G mutant, and many more apoptotic neurons (arrowheads) were observed when both αA456G and βA158W variants were expressed. DNA is in blue. Scale bars represent 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Sieving-defective mutations induce impaired locomotive performance and advanced ageing.(a,b) Negative geotaxis climbing assays were performed to study locomotive behaviour. For each genotype, three groups of male flies were tested at the indicated age. The assay was recorded as video, and the movies were analysed to determine the climbing index. (a) αA456G mutant flies displayed progressively decreasing climbing ability. Expression of the α-subunit (wild-type and αA456G mutant) was driven by Act-Gal4. (b) βA158W did not differ from the wild type during the first 5 weeks. Transgenes were in the β-PheRS background, rescued by gβPheRS or gβA158W under their endogenous promoter. Error bars show s.e.m. n=3, *P<0.05, **P<0.01, t-test. (c,d) Lifespan analysis of the male flies with the same genotype as in a,b. (c) αA456G mutant flies aged faster than wild type, and the differences became apparent already at early stages. P<0.001, log-rank test. (d) At younger age, gβA158W mutant flies aged similarly as the wild type, but at older age (more than 50 days old) the mutants aged faster. P<0.001, log-rank test. (e) Confocal microscopy images of the ventrolateral protocerebrum region of the adult fly brain. Embryos and larvae were kept at 18 °C to keep Gal80ts active to repress Gal4. With this protocol, normal adults eclosed (while the ones expressing αA456G and βA158W were lethal without Gal80ts) and these flies were then incubated at 29 °C for ~7 days, inactivating Gla80ts and activating Gal4. Elav (green) marks neuronal cells, and anti-CP3 stains apoptotic cells (red). A few CP3-positive cells were detected in neurons expressing the αA456G mutant, and many more apoptotic neurons (arrowheads) were observed when both αA456G and βA158W variants were expressed. DNA is in blue. Scale bars represent 5 μm.
Mentions: To further study the long-term effect of PheRS-sd mutations, we analysed their locomotive performance by a negative geotaxis climbing assay. Ubiquitous expression of the wild-type or mutant α-subunit was driven by Act-Gal4, and the climbing capability was recorded once a week (Fig. 4a, Supplementary Movie 1). While αA456G flies showed a similar climbing ability as wild-type animals during the first week, they displayed clearly impaired locomotive performance during the subsequent weeks, and during the 4th week their climbing index decreased to about one quarter of the index of same age control flies. In contrast, the climbing ability of βA158W mutants did not differ significantly from wild-type controls (Fig. 4b). The Act-Gal4-driven expression of the αA456G in the gβA158W transgenic background turned out to be lethal during development, preventing us from examining the locomotive performance of adults.

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