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Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

Feaga HA, Quickel MD, Hankey-Giblin PA, Keiler KC - PLoS Genet. (2016)

Bottom Line: Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA.These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor.Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
Bacteria use trans-translation and the alternative rescue factors ArfA (P36675) and ArfB (Q9A8Y3) to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197) is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

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c. crescentus ArfB rescues human cells from ICT1 silencing.Viability of HEK293 cells expressing ICT1 or ArfB was determined after silencing endogenous ICT1. (A) Western blot showing depletion of ICT1 after silencing with siICT1. Non-targeting siRNA (siNT) was used as a negative control. (B) Schematic diagram showing ArfB with ICT1 localization signal that was used for rescue. (C) Column graphs showing average viable cell numbers from 5 independent experiments. Error bars indicate standard deviation. *** indicates p < 0.0001.
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pgen.1005964.g005: c. crescentus ArfB rescues human cells from ICT1 silencing.Viability of HEK293 cells expressing ICT1 or ArfB was determined after silencing endogenous ICT1. (A) Western blot showing depletion of ICT1 after silencing with siICT1. Non-targeting siRNA (siNT) was used as a negative control. (B) Schematic diagram showing ArfB with ICT1 localization signal that was used for rescue. (C) Column graphs showing average viable cell numbers from 5 independent experiments. Error bars indicate standard deviation. *** indicates p < 0.0001.

Mentions: Mutant versions of ICT1 that lack the GGQ motif required for peptidyl-tRNA hydrolysis do not support viability in human cells when the endogenous ICT1 is silenced [26], indicating that ICT1-mediated peptidyl-tRNA hydrolysis is essential. If the essential activity of ICT1 is rescue of non-stop ribosomes, C. crescentus ArfB might be able to support viability in the absence of ICT1. To test this possibility, a gene encoding the N-terminal sequence of ICT1 containing the mitochondrial localization signal [33] fused to ArfB was constructed. HEK293 cells were transfected with a vector to express ArfB or ICT1, or with an empty vector. Eight hours later, these cells were transfected with siRNA that silenced ICT1 or with control siRNA, and after 6 days the number of living cells was determined. Consistent with previous results, ICT1 silencing decreased the number of viable cells, but cells expressing a version of ICT1 that was not silenced by the siRNA were not affected. Expression of ArfB prior to ICT1 silencing also prevented loss of viability. Cells expressing ArfB prior to ICT1 silencing grew to significantly higher numbers than cells transfected with vector alone (p < 0.0001) (Fig 5). There was no significant difference in viable cell number when cells were rescued with ArfB versus ICT1 (p = 0.75). These results indicate that ArfB can perform the essential function of ICT1 in human cells.


Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

Feaga HA, Quickel MD, Hankey-Giblin PA, Keiler KC - PLoS Genet. (2016)

c. crescentus ArfB rescues human cells from ICT1 silencing.Viability of HEK293 cells expressing ICT1 or ArfB was determined after silencing endogenous ICT1. (A) Western blot showing depletion of ICT1 after silencing with siICT1. Non-targeting siRNA (siNT) was used as a negative control. (B) Schematic diagram showing ArfB with ICT1 localization signal that was used for rescue. (C) Column graphs showing average viable cell numbers from 5 independent experiments. Error bars indicate standard deviation. *** indicates p < 0.0001.
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getmorefigures.php?uid=PMC4814080&req=5

pgen.1005964.g005: c. crescentus ArfB rescues human cells from ICT1 silencing.Viability of HEK293 cells expressing ICT1 or ArfB was determined after silencing endogenous ICT1. (A) Western blot showing depletion of ICT1 after silencing with siICT1. Non-targeting siRNA (siNT) was used as a negative control. (B) Schematic diagram showing ArfB with ICT1 localization signal that was used for rescue. (C) Column graphs showing average viable cell numbers from 5 independent experiments. Error bars indicate standard deviation. *** indicates p < 0.0001.
Mentions: Mutant versions of ICT1 that lack the GGQ motif required for peptidyl-tRNA hydrolysis do not support viability in human cells when the endogenous ICT1 is silenced [26], indicating that ICT1-mediated peptidyl-tRNA hydrolysis is essential. If the essential activity of ICT1 is rescue of non-stop ribosomes, C. crescentus ArfB might be able to support viability in the absence of ICT1. To test this possibility, a gene encoding the N-terminal sequence of ICT1 containing the mitochondrial localization signal [33] fused to ArfB was constructed. HEK293 cells were transfected with a vector to express ArfB or ICT1, or with an empty vector. Eight hours later, these cells were transfected with siRNA that silenced ICT1 or with control siRNA, and after 6 days the number of living cells was determined. Consistent with previous results, ICT1 silencing decreased the number of viable cells, but cells expressing a version of ICT1 that was not silenced by the siRNA were not affected. Expression of ArfB prior to ICT1 silencing also prevented loss of viability. Cells expressing ArfB prior to ICT1 silencing grew to significantly higher numbers than cells transfected with vector alone (p < 0.0001) (Fig 5). There was no significant difference in viable cell number when cells were rescued with ArfB versus ICT1 (p = 0.75). These results indicate that ArfB can perform the essential function of ICT1 in human cells.

Bottom Line: Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA.These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor.Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
Bacteria use trans-translation and the alternative rescue factors ArfA (P36675) and ArfB (Q9A8Y3) to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197) is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

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