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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.

Show MeSH
Drosophila PheRS loci and mutual stabilization of the two subunits.(a) The P{EP}-element insertion allele G2060 is an α-PheRS mutant. (b) β-PheRS1 deletes the first exon of β-PheRS and the neighbouring jar gene. The scheme is on the basis of the information available on FlyBase and is not to scale. (c) Expression of β-PheRS variants in β-PheRS1 and wild-type fly heads. The lower band is wild-type β-PheRS, and the upper band is the myc-tagged β-PheRS. (d) β-PheRS protein levels in Kc cells upon knockdown of the subunits. The control (ctl) is AmpR RNAi. α-PheRS1, α-PheRS2 and α-PheRS3 stand for three different dsRNAs against α-PheRS. Knockdown of either subunit downregulates β-PheRS levels. (e) Myc::α-PheRS protein levels in fly fat bodies upon knockdown of the subunits. ppl-Gal4 drove myc::α-PheRS expression and RNAi against either subunit. The control (ctl) is Cdk7 RNAi. Knockdown of either subunit downregulates Myc::α-PheRS levels. (f) Protein levels upon PheRS overexpression in the fat body dissected from wandering third instar larvae. ppl-Gal4 was used as driver and the control (ctl) is GFP overexpression. Higher levels of protein accumulated only when both subunits (PheRS) were co-overexpressed.
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f1: Drosophila PheRS loci and mutual stabilization of the two subunits.(a) The P{EP}-element insertion allele G2060 is an α-PheRS mutant. (b) β-PheRS1 deletes the first exon of β-PheRS and the neighbouring jar gene. The scheme is on the basis of the information available on FlyBase and is not to scale. (c) Expression of β-PheRS variants in β-PheRS1 and wild-type fly heads. The lower band is wild-type β-PheRS, and the upper band is the myc-tagged β-PheRS. (d) β-PheRS protein levels in Kc cells upon knockdown of the subunits. The control (ctl) is AmpR RNAi. α-PheRS1, α-PheRS2 and α-PheRS3 stand for three different dsRNAs against α-PheRS. Knockdown of either subunit downregulates β-PheRS levels. (e) Myc::α-PheRS protein levels in fly fat bodies upon knockdown of the subunits. ppl-Gal4 drove myc::α-PheRS expression and RNAi against either subunit. The control (ctl) is Cdk7 RNAi. Knockdown of either subunit downregulates Myc::α-PheRS levels. (f) Protein levels upon PheRS overexpression in the fat body dissected from wandering third instar larvae. ppl-Gal4 was used as driver and the control (ctl) is GFP overexpression. Higher levels of protein accumulated only when both subunits (PheRS) were co-overexpressed.

Mentions: Drosophila cytoplasmic PheRS is encoded by the X chromosomal α-PheRS and the third chromosomal β-PheRS. A P-element insertion in the 5′-untranslated repeat of the α-PheRS transcript (Fig. 1a) causes recessive lethality that can be rescued by a genomic copy of α-PheRS (gαPheRS). jar322 deletes the promoter region and the first exon of β-PheRS as well as the neighbouring jar gene27 (Fig. 1b). We named it β-PheRS1 because a genomic copy of β-PheRS (gβPheRS) can rescue its lethality. Rescuing the lethality of hemizygous PheRS1 animals with an myc-tagged genomic copy of β-PheRS (gβPheRS::myc) allowed us to assess levels of PheRS produced from the β-PheRS1 allele. As shown in Fig. 1c, β-PheRS antibodies only detected a single band corresponding to the larger β-PheRS::myc, indicating that β-PheRS1 is indeed a allele. Furthermore, the rescue of the mutant shows that the tagged protein is at least partially functional.


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)

Drosophila PheRS loci and mutual stabilization of the two subunits.(a) The P{EP}-element insertion allele G2060 is an α-PheRS mutant. (b) β-PheRS1 deletes the first exon of β-PheRS and the neighbouring jar gene. The scheme is on the basis of the information available on FlyBase and is not to scale. (c) Expression of β-PheRS variants in β-PheRS1 and wild-type fly heads. The lower band is wild-type β-PheRS, and the upper band is the myc-tagged β-PheRS. (d) β-PheRS protein levels in Kc cells upon knockdown of the subunits. The control (ctl) is AmpR RNAi. α-PheRS1, α-PheRS2 and α-PheRS3 stand for three different dsRNAs against α-PheRS. Knockdown of either subunit downregulates β-PheRS levels. (e) Myc::α-PheRS protein levels in fly fat bodies upon knockdown of the subunits. ppl-Gal4 drove myc::α-PheRS expression and RNAi against either subunit. The control (ctl) is Cdk7 RNAi. Knockdown of either subunit downregulates Myc::α-PheRS levels. (f) Protein levels upon PheRS overexpression in the fat body dissected from wandering third instar larvae. ppl-Gal4 was used as driver and the control (ctl) is GFP overexpression. Higher levels of protein accumulated only when both subunits (PheRS) were co-overexpressed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Drosophila PheRS loci and mutual stabilization of the two subunits.(a) The P{EP}-element insertion allele G2060 is an α-PheRS mutant. (b) β-PheRS1 deletes the first exon of β-PheRS and the neighbouring jar gene. The scheme is on the basis of the information available on FlyBase and is not to scale. (c) Expression of β-PheRS variants in β-PheRS1 and wild-type fly heads. The lower band is wild-type β-PheRS, and the upper band is the myc-tagged β-PheRS. (d) β-PheRS protein levels in Kc cells upon knockdown of the subunits. The control (ctl) is AmpR RNAi. α-PheRS1, α-PheRS2 and α-PheRS3 stand for three different dsRNAs against α-PheRS. Knockdown of either subunit downregulates β-PheRS levels. (e) Myc::α-PheRS protein levels in fly fat bodies upon knockdown of the subunits. ppl-Gal4 drove myc::α-PheRS expression and RNAi against either subunit. The control (ctl) is Cdk7 RNAi. Knockdown of either subunit downregulates Myc::α-PheRS levels. (f) Protein levels upon PheRS overexpression in the fat body dissected from wandering third instar larvae. ppl-Gal4 was used as driver and the control (ctl) is GFP overexpression. Higher levels of protein accumulated only when both subunits (PheRS) were co-overexpressed.
Mentions: Drosophila cytoplasmic PheRS is encoded by the X chromosomal α-PheRS and the third chromosomal β-PheRS. A P-element insertion in the 5′-untranslated repeat of the α-PheRS transcript (Fig. 1a) causes recessive lethality that can be rescued by a genomic copy of α-PheRS (gαPheRS). jar322 deletes the promoter region and the first exon of β-PheRS as well as the neighbouring jar gene27 (Fig. 1b). We named it β-PheRS1 because a genomic copy of β-PheRS (gβPheRS) can rescue its lethality. Rescuing the lethality of hemizygous PheRS1 animals with an myc-tagged genomic copy of β-PheRS (gβPheRS::myc) allowed us to assess levels of PheRS produced from the β-PheRS1 allele. As shown in Fig. 1c, β-PheRS antibodies only detected a single band corresponding to the larger β-PheRS::myc, indicating that β-PheRS1 is indeed a allele. Furthermore, the rescue of the mutant shows that the tagged protein is at least partially functional.

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