Limits...
A translation system reconstituted with human factors proves that processing of encephalomyocarditis virus proteins 2A and 2B occurs in the elongation phase of translation without eukaryotic release factors.

Machida K, Mikami S, Masutani M, Mishima K, Kobayashi T, Imataka H - J. Biol. Chem. (2014)

Bottom Line: These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event.We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs.Our results indicate that this unusual event occurs in the elongation phase of translation.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Materials Science and Chemistry and Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan and.

Show MeSH

Related in: MedlinePlus

2A is released from tRNA. The HeLa cell extract-derived protein expression system was programmed with pUC-T7-HCV HA-2A-2B-FLAG for lanes 1, 3, and 5 or with pUC-T7-HCV HA-2A(uORF2) for lanes 2, 4, and 6. After translation (10 min), samples were treated with EDTA (19.2 mm) for 5 min at 25 °C and then supplemented with magnesium acetate (22.3 mm). Each sample was subsequently treated with water (lanes 1 and 2), RNase A (0.1 μg/μl) (lanes 3 and 4), or peptidyl-tRNA hydrolase (PTH) (0.42 μg/μl) (lanes 5 and 6) for 30 min at 25 °C and resolved by NuPAGE followed by Western blotting (WB) with anti-HA antibody. The arrowhead indicates the band that disappeared due to RNase or peptidyl-tRNA hydrolase treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231674&req=5

Figure 3: 2A is released from tRNA. The HeLa cell extract-derived protein expression system was programmed with pUC-T7-HCV HA-2A-2B-FLAG for lanes 1, 3, and 5 or with pUC-T7-HCV HA-2A(uORF2) for lanes 2, 4, and 6. After translation (10 min), samples were treated with EDTA (19.2 mm) for 5 min at 25 °C and then supplemented with magnesium acetate (22.3 mm). Each sample was subsequently treated with water (lanes 1 and 2), RNase A (0.1 μg/μl) (lanes 3 and 4), or peptidyl-tRNA hydrolase (PTH) (0.42 μg/μl) (lanes 5 and 6) for 30 min at 25 °C and resolved by NuPAGE followed by Western blotting (WB) with anti-HA antibody. The arrowhead indicates the band that disappeared due to RNase or peptidyl-tRNA hydrolase treatment.

Mentions: The 2A fragment might be generated due to a translational pause of the ribosome at the C terminus of the 2A region. In that case, the 2A protein should remain ligated to the tRNAGly in the ribosomal P site as demonstrated in SecM-mediated ribosomal stall (31), translational pause in the XBP1u mRNA (32), and ribosomal arrest mediated by the upstream open reading frame 2 (uORF2) of the cytomegalovirus UL 4 gene (33). To examine this, we incubated the HeLa cell extract with the HA-2A-2B-FLAG construct and resolved the product with NuPAGE (Invitrogen; the pH of the gel is neutral) (34) to maximally retain the esterified bond between the 3′-end of the tRNA and the C terminus of the 2A peptide. Western blotting with anti-HA antibody detected the 2A peptide (Fig. 3, lane 1). We tested a positive control with a plasmid encoding HA-2A(uORF2) in which the C-terminal 22 amino acids of 2A had been replaced with uORF2 of the cytomegalovirus UL 4 gene (33). From this template, a product that migrated slower than the HA-2A peptide was generated (lane 2). Treatment with RNase A eliminated this slowly migrating product (lane 4). This product was also abolished by treatment with peptidyl-tRNA hydrolase (Ref. 35; kindly provided by Dr. Nagao) (we confirmed that the peptidyl-tRNA hydrolase preparation used in this experiment was devoid of an RNase activity that would decompose the body of tRNA; data not shown), indicating that the peptide was conjugated to tRNA. From the HA-2A-2B-FLAG construct, neither RNase A- nor peptidyl-tRNA hydrolase-sensitive product was produced (Fig. 3, lanes 1, 3, and 5). Thus, the 2A peptide was dissociated from tRNA despite the absence of a termination codon.


A translation system reconstituted with human factors proves that processing of encephalomyocarditis virus proteins 2A and 2B occurs in the elongation phase of translation without eukaryotic release factors.

Machida K, Mikami S, Masutani M, Mishima K, Kobayashi T, Imataka H - J. Biol. Chem. (2014)

2A is released from tRNA. The HeLa cell extract-derived protein expression system was programmed with pUC-T7-HCV HA-2A-2B-FLAG for lanes 1, 3, and 5 or with pUC-T7-HCV HA-2A(uORF2) for lanes 2, 4, and 6. After translation (10 min), samples were treated with EDTA (19.2 mm) for 5 min at 25 °C and then supplemented with magnesium acetate (22.3 mm). Each sample was subsequently treated with water (lanes 1 and 2), RNase A (0.1 μg/μl) (lanes 3 and 4), or peptidyl-tRNA hydrolase (PTH) (0.42 μg/μl) (lanes 5 and 6) for 30 min at 25 °C and resolved by NuPAGE followed by Western blotting (WB) with anti-HA antibody. The arrowhead indicates the band that disappeared due to RNase or peptidyl-tRNA hydrolase treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231674&req=5

Figure 3: 2A is released from tRNA. The HeLa cell extract-derived protein expression system was programmed with pUC-T7-HCV HA-2A-2B-FLAG for lanes 1, 3, and 5 or with pUC-T7-HCV HA-2A(uORF2) for lanes 2, 4, and 6. After translation (10 min), samples were treated with EDTA (19.2 mm) for 5 min at 25 °C and then supplemented with magnesium acetate (22.3 mm). Each sample was subsequently treated with water (lanes 1 and 2), RNase A (0.1 μg/μl) (lanes 3 and 4), or peptidyl-tRNA hydrolase (PTH) (0.42 μg/μl) (lanes 5 and 6) for 30 min at 25 °C and resolved by NuPAGE followed by Western blotting (WB) with anti-HA antibody. The arrowhead indicates the band that disappeared due to RNase or peptidyl-tRNA hydrolase treatment.
Mentions: The 2A fragment might be generated due to a translational pause of the ribosome at the C terminus of the 2A region. In that case, the 2A protein should remain ligated to the tRNAGly in the ribosomal P site as demonstrated in SecM-mediated ribosomal stall (31), translational pause in the XBP1u mRNA (32), and ribosomal arrest mediated by the upstream open reading frame 2 (uORF2) of the cytomegalovirus UL 4 gene (33). To examine this, we incubated the HeLa cell extract with the HA-2A-2B-FLAG construct and resolved the product with NuPAGE (Invitrogen; the pH of the gel is neutral) (34) to maximally retain the esterified bond between the 3′-end of the tRNA and the C terminus of the 2A peptide. Western blotting with anti-HA antibody detected the 2A peptide (Fig. 3, lane 1). We tested a positive control with a plasmid encoding HA-2A(uORF2) in which the C-terminal 22 amino acids of 2A had been replaced with uORF2 of the cytomegalovirus UL 4 gene (33). From this template, a product that migrated slower than the HA-2A peptide was generated (lane 2). Treatment with RNase A eliminated this slowly migrating product (lane 4). This product was also abolished by treatment with peptidyl-tRNA hydrolase (Ref. 35; kindly provided by Dr. Nagao) (we confirmed that the peptidyl-tRNA hydrolase preparation used in this experiment was devoid of an RNase activity that would decompose the body of tRNA; data not shown), indicating that the peptide was conjugated to tRNA. From the HA-2A-2B-FLAG construct, neither RNase A- nor peptidyl-tRNA hydrolase-sensitive product was produced (Fig. 3, lanes 1, 3, and 5). Thus, the 2A peptide was dissociated from tRNA despite the absence of a termination codon.

Bottom Line: These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event.We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs.Our results indicate that this unusual event occurs in the elongation phase of translation.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Materials Science and Chemistry and Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji 671-2201, Japan and.

Show MeSH
Related in: MedlinePlus