A PABPC1 mutation disturbing its association with eIF4G reduces its capacity to suppress NMD.

<p>(A) HeLa cells were transiently transfected with the NMD reporter miniμ ter310 construct A or construct B, plasmids encoding the indicated PABPC1-MS2 fusion protein variants and GPx1 as a normalizer. After RNA extraction and RT-qPCR, relative miniμ ter310 and GPx1 mRNA were measured and normalized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g001" target="_blank"><b>Figure 1C</b></a>, except that the LacZ-MS2 samples were set as 100%. Full length PABPC1 (1–636-MS2) and a version comprising the first four RNA recognition motifs (1–372-MS2) with or without the M161A mutation are shown. All proteins are fused to the N-terminus of the MS2 moiety and contain a HA-tag at the C-terminus. Average values and standard deviations of at least three independent experiments are shown. The right panel shows a western blot using anti-HA and anti-tubulin antibodies to monitor the expression levels of the transfected MS2 fusion proteins and the endogenous tubulin as loading control, respectively. (B) To assess the effect of the M161A mutation in PABPC1 on the interaction with eIF4G, HEK 293T cells were transfected with plasmids encoding the eIF4GI isoform e fused to MS2 (eIF4Gle-MS2; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g004" target="_blank"><b>Figure 4</b></a>) or LacZ-MS2 together with a plasmid encoding the indicated PABPC1 construct. All proteins were HA-tagged. 48 h post transfection, immunoprecipitations were performed using an anti-MS2 antibody and the association of the PABPC1 constructs with eIF4Gle was assessed by western blotting using an anti-HA antibody. Samples before (input) and the supernatant after (unbound) the immunoprecipitations represent 10% of the total material, and 50% of the immunoprecipitated material (IP) were loaded on the gel.</p

Tethering of the core domain of eIF4GI inhibits NMD to a similar extent as PABPC1.

<p>(A) Schematic representations of the eukaryotic initiation factor 4GI (eIF4GI; adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone.0104391-Coldwell1" target="_blank">[53]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone.0104391-Marintchev1" target="_blank">[80]</a>) and the CBP80/20-dependent translation initiation factor (CTIF; adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone.0104391-Kim1" target="_blank">[57]</a>). Functional domains are color-coded and labeled (RRM, RNA recognition motif; MIF4G, middle domain of eIF4G and HEAT-1 domain; MA3, HEAT-2 domain and MA3 region; W2, HEAT-3 and W2 domain). The letters (f, e, d, b, a) indicate the different N-termini of these five eIF4GI isoforms. Numbers below represent the respective amino acid positions, with 1 depicting the N-terminus of the longest eIF4GI isoform, eIF4Gf. Isoform c, which is 1 amino acid shorter than isoform d, is not shown. (B) Tethering of the different eIF4GI isoforms shown schematically on the right (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g004" target="_blank"><b>Figure 4A</b></a>). HeLa cells were transiently transfected with the NMD reporter miniμ ter310 construct A or construct B, a plasmid encoding the indicated eIF4GI-MS2 fusion protein and one encoding GPx1 for normalization. The assay was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g001" target="_blank"><b>Figure 1C</b></a>. Average miniμ mRNA levels and standard deviations of at least four independent experiments, normalized to GPx1 mRNA and displayed relative to the LacZ-MS2 samples are shown, except for eIF4Gld and eIF4Glb where the results of only one experiment are shown. Full length PABPC1-MS2 corresponds to 1–636-MS2 in previous Figures. All MS2 fusion proteins contain a C-terminal HA-tag. The lower panel represents a western blot probed with anti-HA and anti-Tubulin antibodies to assess the relative expression of the MS2-fusion proteins and of endogenous tubulin used as a loading control, respectively. (C) To test for association of endogenous PABPC1 with eIF4GI variants, plasmids encoding the indicated eIF4GI isoforms or the eIF4GI core domain (eIF4GI682-1130-MS2) fused to MS2-HA were transfected into HEK 293T cells. After 48 h, the cell extracts were subjected to immunoprecipitation using an anti-MS2 antibody. LacZ-MS2 expressing cells served as a specificity control. The MS2 fusion proteins were detected with an antibody against the HA-tag (upper panels) and endogenous PABPC1 was detected with the mouse anti-PABPC1 10E10 antibody (lower panels). 10% of the cell extracts before (input) and the supernatant after (unbound) the immunoprecipitations, and 50% of the immunoprecipitated material (IP) were loaded on the gel. (D) Tethering assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g001" target="_blank"><b>Figure 1C</b></a>, but with the eIF4GI deletion mutants depicted schematically on the right. All MS2 fusion proteins also contain a C-terminal HA-tag. Lower panel, western blot as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g004" target="_blank"><b>Figure 4B</b></a>. (E) Tethering assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g001" target="_blank"><b>Figure 1C</b></a>, but with CTIF-MS2. Average values and standard deviations of three independent experiments are shown. The western blot shown on the right side was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104391#pone-0104391-g004" target="_blank"><b>Figure 4B</b></a>.</p