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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 mutant flies are sensitive to non-cognate amino acids.(a) Scheme illustrating the experimental procedure. The ppl-Gal4 driver was used to drive PheRS-sd mutations in the larval fat body. Embryos were aged to 24 h after egg laying. First instar larvae with the correct genotype were selected and transferred to different amino-acid-rich food. When the larvae developed into pupae, the number of pupating individuals was recorded to calculate the pupariation rate. (b–e) PheRS-sd mutant flies are sensitive to the non-cognate amino acid Tyr. Larvae were raised on standard food (b), Ala-rich food (100 mM, c), Phe-rich food (100 mM, d) and Tyr-rich food (100 mM, e). No significant differences in pupariation rate were observed between control and PheRS-sd mutants on standard food and Ala- or Phe-rich food. On Tyr-rich food, the pupariation rate of the αA456G mutant was reduced to ~60%, and the one of larvae expressing αA456G and βA158W PheRS to ~30%. Error bars represent s.e.m. n=6. **P<0.01, *P<0.05, t-test.
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f8: PheRS-sd mutant flies are sensitive to non-cognate amino acids.(a) Scheme illustrating the experimental procedure. The ppl-Gal4 driver was used to drive PheRS-sd mutations in the larval fat body. Embryos were aged to 24 h after egg laying. First instar larvae with the correct genotype were selected and transferred to different amino-acid-rich food. When the larvae developed into pupae, the number of pupating individuals was recorded to calculate the pupariation rate. (b–e) PheRS-sd mutant flies are sensitive to the non-cognate amino acid Tyr. Larvae were raised on standard food (b), Ala-rich food (100 mM, c), Phe-rich food (100 mM, d) and Tyr-rich food (100 mM, e). No significant differences in pupariation rate were observed between control and PheRS-sd mutants on standard food and Ala- or Phe-rich food. On Tyr-rich food, the pupariation rate of the αA456G mutant was reduced to ~60%, and the one of larvae expressing αA456G and βA158W PheRS to ~30%. Error bars represent s.e.m. n=6. **P<0.01, *P<0.05, t-test.

Mentions: Because of its similar structure Tyr, can be misactivated by PheRS. Expecting that PheRS-sd mutations increase the production of misacylated Tyr-tRNAPhe, we tested whether these mutations are sensitive to Tyr. Using ppl-Gal4 we expressed the PheRS-sd mutations in the fat body, a tissue that has a high demand for nutrients and grows dramatically at the larval stage39. Larvae were challenged by the addition of different amino acids to the fly food and their pupariation rate was recorded (Fig. 8a). Reared on normal fly food, there was no difference in the pupariation rate between the wild type, αA456G-expressing larvae and larvae expressing mutant αA456G plus βA158W PheRS (Fig. 8b). In addition, no significant differences were observed between the Ala-rich and Phe-rich food (Fig. 8c,d). In contrast, Tyr-rich food reduced the pupariation rate of αA456G-expressing larvae to ~60%, and the rate of larvae expressing both mutant forms to ~30% of the wild type (Fig. 8e). This specific sensitivity suggests that these PheRS mutations are sieving-defective in vivo.


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 mutant flies are sensitive to non-cognate amino acids.(a) Scheme illustrating the experimental procedure. The ppl-Gal4 driver was used to drive PheRS-sd mutations in the larval fat body. Embryos were aged to 24 h after egg laying. First instar larvae with the correct genotype were selected and transferred to different amino-acid-rich food. When the larvae developed into pupae, the number of pupating individuals was recorded to calculate the pupariation rate. (b–e) PheRS-sd mutant flies are sensitive to the non-cognate amino acid Tyr. Larvae were raised on standard food (b), Ala-rich food (100 mM, c), Phe-rich food (100 mM, d) and Tyr-rich food (100 mM, e). No significant differences in pupariation rate were observed between control and PheRS-sd mutants on standard food and Ala- or Phe-rich food. On Tyr-rich food, the pupariation rate of the αA456G mutant was reduced to ~60%, and the one of larvae expressing αA456G and βA158W PheRS to ~30%. Error bars represent s.e.m. n=6. **P<0.01, *P<0.05, t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: PheRS-sd mutant flies are sensitive to non-cognate amino acids.(a) Scheme illustrating the experimental procedure. The ppl-Gal4 driver was used to drive PheRS-sd mutations in the larval fat body. Embryos were aged to 24 h after egg laying. First instar larvae with the correct genotype were selected and transferred to different amino-acid-rich food. When the larvae developed into pupae, the number of pupating individuals was recorded to calculate the pupariation rate. (b–e) PheRS-sd mutant flies are sensitive to the non-cognate amino acid Tyr. Larvae were raised on standard food (b), Ala-rich food (100 mM, c), Phe-rich food (100 mM, d) and Tyr-rich food (100 mM, e). No significant differences in pupariation rate were observed between control and PheRS-sd mutants on standard food and Ala- or Phe-rich food. On Tyr-rich food, the pupariation rate of the αA456G mutant was reduced to ~60%, and the one of larvae expressing αA456G and βA158W PheRS to ~30%. Error bars represent s.e.m. n=6. **P<0.01, *P<0.05, t-test.
Mentions: Because of its similar structure Tyr, can be misactivated by PheRS. Expecting that PheRS-sd mutations increase the production of misacylated Tyr-tRNAPhe, we tested whether these mutations are sensitive to Tyr. Using ppl-Gal4 we expressed the PheRS-sd mutations in the fat body, a tissue that has a high demand for nutrients and grows dramatically at the larval stage39. Larvae were challenged by the addition of different amino acids to the fly food and their pupariation rate was recorded (Fig. 8a). Reared on normal fly food, there was no difference in the pupariation rate between the wild type, αA456G-expressing larvae and larvae expressing mutant αA456G plus βA158W PheRS (Fig. 8b). In addition, no significant differences were observed between the Ala-rich and Phe-rich food (Fig. 8c,d). In contrast, Tyr-rich food reduced the pupariation rate of αA456G-expressing larvae to ~60%, and the rate of larvae expressing both mutant forms to ~30% of the wild type (Fig. 8e). This specific sensitivity suggests that these PheRS mutations are sieving-defective in vivo.

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