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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 ER stress.An Xbp1-eGFP reporter was used to monitor the expression of the spliced Xbp1 isoform in PheRS-sd mutant wing discs using immunostaining. While no signal was observed in controls, GFP expression was detected both in αA456G mutants and in cells expressing the mutant forms αA456G and βA158W. Note that the GFP signal is always restricted to the posterior compartment. DNA is in blue and GFP is in green. Scale bars represent 25 μm.
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f7: Sieving-defective mutations induce ER stress.An Xbp1-eGFP reporter was used to monitor the expression of the spliced Xbp1 isoform in PheRS-sd mutant wing discs using immunostaining. While no signal was observed in controls, GFP expression was detected both in αA456G mutants and in cells expressing the mutant forms αA456G and βA158W. Note that the GFP signal is always restricted to the posterior compartment. DNA is in blue and GFP is in green. Scale bars represent 25 μm.

Mentions: The PheRS αA456G mutation is predicted to increase non-cognate amino-acid activation, and the βA158W mutant is not able to edit the faulty activation. As a consequence, non-cognate amino acids could be incorporated during protein synthesis, leading to the accumulation of misfolded proteins. In the lumen of the ER, misfolded proteins can impair the protein-folding capacity of the ER, a condition known as ER stress353637. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response including cytoplasmic Xbp1 mRNA splicing. To test whether the PheRS-sd mutations induce ER stress, we monitored Xbp1 splicing with the Xbp1-eGFP (enhanced GFP) reporter38, in which the splicing-induced frameshift leads to eGFP expression. Wing discs expressing the αA456G mutant form or the αA456G and βA158W mutant forms of PheRS in the posterior compartment showed clear GFP expression above control levels in this compartment (Fig. 7). We also stained for the ER stress marker phosphorylated eukaryotic initiation factor 2α. Immunofluorescence analysis demonstrated that eukaryotic initiation factor 2α is transiently phosphorylated in several wing discs expressing both mutant forms together, but not in the ones expressing only the mutant α-PheRS αA456G in the posterior compartment or only the mutant β-PheRS in the anterior compartment (Supplementary Fig. 2). These results together revealed that the defect in the first sieve causes a certain level of ER stress and defects in both sieves induce strong ER stress.


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 ER stress.An Xbp1-eGFP reporter was used to monitor the expression of the spliced Xbp1 isoform in PheRS-sd mutant wing discs using immunostaining. While no signal was observed in controls, GFP expression was detected both in αA456G mutants and in cells expressing the mutant forms αA456G and βA158W. Note that the GFP signal is always restricted to the posterior compartment. DNA is in blue and GFP is in green. Scale bars represent 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Sieving-defective mutations induce ER stress.An Xbp1-eGFP reporter was used to monitor the expression of the spliced Xbp1 isoform in PheRS-sd mutant wing discs using immunostaining. While no signal was observed in controls, GFP expression was detected both in αA456G mutants and in cells expressing the mutant forms αA456G and βA158W. Note that the GFP signal is always restricted to the posterior compartment. DNA is in blue and GFP is in green. Scale bars represent 25 μm.
Mentions: The PheRS αA456G mutation is predicted to increase non-cognate amino-acid activation, and the βA158W mutant is not able to edit the faulty activation. As a consequence, non-cognate amino acids could be incorporated during protein synthesis, leading to the accumulation of misfolded proteins. In the lumen of the ER, misfolded proteins can impair the protein-folding capacity of the ER, a condition known as ER stress353637. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response including cytoplasmic Xbp1 mRNA splicing. To test whether the PheRS-sd mutations induce ER stress, we monitored Xbp1 splicing with the Xbp1-eGFP (enhanced GFP) reporter38, in which the splicing-induced frameshift leads to eGFP expression. Wing discs expressing the αA456G mutant form or the αA456G and βA158W mutant forms of PheRS in the posterior compartment showed clear GFP expression above control levels in this compartment (Fig. 7). We also stained for the ER stress marker phosphorylated eukaryotic initiation factor 2α. Immunofluorescence analysis demonstrated that eukaryotic initiation factor 2α is transiently phosphorylated in several wing discs expressing both mutant forms together, but not in the ones expressing only the mutant α-PheRS αA456G in the posterior compartment or only the mutant β-PheRS in the anterior compartment (Supplementary Fig. 2). These results together revealed that the defect in the first sieve causes a certain level of ER stress and defects in both sieves induce strong ER stress.

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