Figure 3. Effect of EDF1 on frameshifting at stall sites.

(A) Schematic representation of the reporter constructs used to analyse frameshifting. RFP was directly appended to each of the three tested XBP1 reporters (see amino acid sequences below) in three different reading frames. Note that S255A mutation exacerbates, whereas W256A reduces ribosomal stalling, relative to the wild type XBP1 sequence. (B) In-frame versions of the constructs from panel A were co-transfected with BFP-encoding plasmid into Flp-In T-REx 293 cells and analysed by flow cytometry after 20 hr of transgene expression. At least 20,000 transfected (BFP-positive) cells were analysed here and in all the subsequent experiments involving frameshifting reporters. The RFP:GFP ratio, an indicator of readthrough at the XBP1 sequence, is plotted as a histogram for each reporter. (C) The same in-frame constructs as in panel B were analysed in WT and ZNF598 KO cells. Note that the RFP:GFP ratio is elevated in ∆ZNF598 cells for WT XBP1 and XBP1(S255A), but not for XBP1(W256A). (D) XBP1(S255A) (left) or XBP1(W256A) (right) was transfected into cells pre-treated for 72 hr with non-targeting siRNA (control) or EDF1-targeting siRNA and analysed by flow cytometry after 20 hr of reporter expression. Expression of GFP (bottom panels) and RFP (top panels) was plotted as histograms. Gray shaded traces represent cells positively transfected with in-frame (+0) constructs, blue traces represent −1 constructs and red traces represent +1 constructs. Background fluorescence observed in non-transfected cells is indicated by the unshaded thin gray traces. The amount of RFP signal observed for out-of-frame constructs (+1,–1), represented as a percentage of the in-frame signal, is indicated above each graph. Note that GFP expression is not influenced by the RFP reading frame.

Figure 3—figure supplement 1. Effect of EDF1 on frameshifting at stall sites.

(A) Schematic representation of the reporter constructs used to analyse frameshifting. RFP was directly appended to (K^AAA)₁₂ stalling sequence in three different reading frames. (B) Constructs from panel A were transfected into cells pre-treated for 72 hr with non-targeting siRNA (control) or EDF1-targeting siRNA and analysed by flow cytometry after 20 hr of reporter expression. Expression of GFP (bottom panels) and RFP (top panels) was plotted as histograms. Gray shaded traces represent cells positively transfected with in-frame (+0) constructs, blue traces represent −1 constructs and red traces represent +1 constructs. Background fluorescence observed in non-transfected cells is indicated by the unshaded thin gray traces. The amount of RFP signal observed for out-of-frame constructs (+1,–1), represented as a percentage of the in-frame signal, is indicated above each graph. Note that GFP expression is not influenced by the RFP reading frame. (C) Total cell lysates from cells depleted for EDF1 using two different siRNA sequences were analysed by western blotting alongside the lysates from control siRNA-treated and non-treated cells. The siRNA treatment here and in other experiments which used siRNA-mediated depletion of EDF1 was for 72 hr.

Figure 3—figure supplement 2. Effect of EDF1 on frameshifting at stall sites.

(A) Analysis of the frameshifting reporters containing WT XBP1 exactly as in Figure 3D. (B) Analysis of the frameshifting reporters containing XBP1(S255A) as in Figure 3D using two different siRNAs targeting EDF1. In both panel A and panel B, the reporter constructs were transfected into cells pre-treated for 72 hr with non-targeting siRNA (control) or EDF1-targeting siRNA and analysed by flow cytometry after 20 hr of reporter expression. Expression of GFP (bottom panels) and RFP (top panels) was plotted as histograms. Gray shaded traces represent cells positively transfected with in-frame (+0) constructs, blue traces represent −1 constructs and red traces represent +1 constructs. Background fluorescence observed in non-transfected cells is indicated by the unshaded thin gray traces. The amount of RFP signal observed for out-of-fram constructs (+1,–1), represented as a percentage of the in-frame signal, is indicated above each graph. Note that GFP expression is not influenced by the RFP reading frame.