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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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Related in: MedlinePlus

ICT1 and ArfB hydrolyze peptidyl-tRNA on ribosomes near the 3’ end of an mRNA.In vitro transcription/translation reactions were performed with template lacking a stop codon, or with template with 0, 6, 14, or 33 bases past the stop codon. (A) Cartoons depicting the expected result of translation in the absence of added rescue or release factors. (B) Representative autoradiograms of reactions resolved on Bis-Tris gels. (C) Column graphs show average release activity from ≥3 replicates with error bars indicating the standard deviation.
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pgen.1005964.g002: ICT1 and ArfB hydrolyze peptidyl-tRNA on ribosomes near the 3’ end of an mRNA.In vitro transcription/translation reactions were performed with template lacking a stop codon, or with template with 0, 6, 14, or 33 bases past the stop codon. (A) Cartoons depicting the expected result of translation in the absence of added rescue or release factors. (B) Representative autoradiograms of reactions resolved on Bis-Tris gels. (C) Column graphs show average release activity from ≥3 replicates with error bars indicating the standard deviation.

Mentions: To confirm that ICT1 can hydrolyze peptidyl-tRNA on non-stop ribosomes in a manner similar to ArfB, a coupled transcription-translation system lacking RF1, RF2, and RF3 was used to express a folA gene (encoding DHFR) that lacked a stop codon. Consistent with previous observations of peptidyl-tRNA hydrolysis on non-stop ribosomes [15,16,21], addition of a release factor mixture containing RF-1, RF-2, and RF-3 to the reaction had little effect on the percentage of DHFR found in peptidyl-tRNA, but when ArfB was included 74 ± 1% of the DHFR was released (Fig 2). When ICT1 was included in the reaction, 78 ± 8% DHFR was released, indicating that ICT1 has a similar activity to ArfB on non-stop ribosomes.


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

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

ICT1 and ArfB hydrolyze peptidyl-tRNA on ribosomes near the 3’ end of an mRNA.In vitro transcription/translation reactions were performed with template lacking a stop codon, or with template with 0, 6, 14, or 33 bases past the stop codon. (A) Cartoons depicting the expected result of translation in the absence of added rescue or release factors. (B) Representative autoradiograms of reactions resolved on Bis-Tris gels. (C) Column graphs show average release activity from ≥3 replicates with error bars indicating the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814080&req=5

pgen.1005964.g002: ICT1 and ArfB hydrolyze peptidyl-tRNA on ribosomes near the 3’ end of an mRNA.In vitro transcription/translation reactions were performed with template lacking a stop codon, or with template with 0, 6, 14, or 33 bases past the stop codon. (A) Cartoons depicting the expected result of translation in the absence of added rescue or release factors. (B) Representative autoradiograms of reactions resolved on Bis-Tris gels. (C) Column graphs show average release activity from ≥3 replicates with error bars indicating the standard deviation.
Mentions: To confirm that ICT1 can hydrolyze peptidyl-tRNA on non-stop ribosomes in a manner similar to ArfB, a coupled transcription-translation system lacking RF1, RF2, and RF3 was used to express a folA gene (encoding DHFR) that lacked a stop codon. Consistent with previous observations of peptidyl-tRNA hydrolysis on non-stop ribosomes [15,16,21], addition of a release factor mixture containing RF-1, RF-2, and RF-3 to the reaction had little effect on the percentage of DHFR found in peptidyl-tRNA, but when ArfB was included 74 ± 1% of the DHFR was released (Fig 2). When ICT1 was included in the reaction, 78 ± 8% DHFR was released, indicating that ICT1 has a similar activity to ArfB on non-stop ribosomes.

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.

Show MeSH
Related in: MedlinePlus