Proteasomal degradation of human release factor eRF3a regulates translation termination complex formation

Abstract

In eukaryotes, eRF1 and eRF3 are associated in a complex that mediates translation termination. The regulation of the formation of this complex in vivo is far from being understood. In mammalian cells, depletion of eRF3a causes a reduction of eRF1 level by decreasing its stability. Here, we investigate the status of eRF3a when not associated with eRF1. We show that eRF3a forms altered in their eRF1-binding site have a decreased stability, which increases upon cell treatment with the proteasome inhibitor MG132. We also show that eRF3a forms altered in eRF1 binding as well as wild-type eRF3a are polyubiquitinated. These results indicate that eRF3a is degraded by the proteasome when not associated with eRF1 and suggest that proteasomal degradation of eRF3a controls translation termination complex formation by adjusting the eRF3a level to that of eRF1.

FIGURE 1.

Stabilization of eRF3a forms mutated in eRF1-binding site by MG132. Extracts of HEK293 cells electroporated with (A) the empty vector pBK-CMV (lanes pBK), plasmid expressing wild-type human eRF3a (lanes 3a), plasmids expressing eRF3a variants with either a double mutation in eRF1-binding site (lanes FRAA), or a deletion of the last 31 amino acids containing the eRF1-binding site (lanes ΔC); (B) plasmids expressing GFP fusion with wild-type eRF3a (lanes GFP-3a), and with eRF3a variant carrying the double mutation in the eRF1-binding site (lanes GFP-3a-FRAA) were analyzed by Western blotting with antibodies to human eRF3a; equal protein loading was verified by the detection of α-tubulin (α-tub). Cells were treated or not with MG132 as indicated above each lane. (C) Translation termination activity of eRF3a forms mutated in the eRF1-binding site. 559C cells stably expressing a lacZ gene containing a premature stop codon were depleted in eRF3a by electroporation of a plasmid expressing a siRNA (si-3a1) directed against eRF3a mRNA and re-electroporated 3 d later with either the empty vector pBK-CMV (3a1/pBK) or plasmid expressing wild-type eRF3a (3a1/3a), or plasmids expressing the variants eRF3a-FRAA (3a1/FRAA), eRF3a-ΔC (3a1/ΔC), GFP-eRF3a (3a1/GFP-3a), and GFP-3a-FRAA (3a1/GFP-3a-FRAA). Cells were treated or not with MG132 as indicated, and the extracts performed 3 d after the second electroporation were used for the β-galactosidase assay. The results of readthrough assays were normalized to the level of 3a1/pBK-CMV electroporated cells, which served as the standard for the readthrough level promoted by eRF3a depletion (lane 3a1/pBK). Thus, for each experiment and for each cell culture condition, the percent readthrough level relative to the readthrough level of cells electroporated with plasmid expressing si-3a1 and re-electroporated with pBK-CMV (3a1/pBK) was calculated and expressed as the readthrough efficiency. Results were expressed as the mean of three experiments; error bars show the standard error of the mean.

FIGURE 2.

Ubiquitination of GFP-3a and GFP-3a-FRAA. (A) HEK293 cells expressing GFP-3a (lanes GFP-3a) and GFP-3a-FRAA (lanes GFP-3a-FRAA) fusion proteins were treated with MG132, and proteins (200 μg) were immunoprecipitated with mouse monoclonal anti-GFP antibodies. Protein input (20 μg) and immunoprecipitates (IP:GFP) were analyzed by Western blotting using anti-eRF3a (top panel) and anti-eRF1 (bottom panel) rabbit polyclonal antibodies. (B) HEK293 cells expressing GFP-3a (lanes GFP-3a) and GFP-3a-FRAA (lanes GFP-3a-FRAA) fusion proteins were treated or not with MG132 as indicated above each lane. Cell extracts (200 μg of protein) were immunoprecipitated with mouse monoclonal anti-GFP antibodies, and immunoprecipitates were analyzed by Western blotting using anti-eRF3a (left panel) and anti-ubiquitin (right panel) antibodies.

FIGURE 3.

Pulse-chase labeling of HEK293 cells expressing (A) GFP-3a and (B) GFP-3a-FRAA. Labeled proteins were immunoprecipitated with anti-GFP (top panel) and anti-α-tubulin antibodies (bottom panel). (C) [³⁵S]-Labeled GFP-3a and GFP-3a-FRAA bands were quantified using the BAS1000 Fuji image plate program V 2.0, and the value at each time point was plotted against time: (filled triangles) GFP-3A, (filled squares) GFP-3a-FRAA. The regression line is presented for each curve.

FIGURE 4.

A model for the regulation of the translation termination complex formation (see description in the Discussion).