Figure 3. Antisense C₄G₂ repeat expanded RNAs activate the PKR/eIF2α pathway independent of DPR proteins.

(A) (Top) Schematic illustration of (C₄G₂)75 repeats without the human intronic sequences. 3× protein tags were included at the C- terminus of the repeats to monitor the DPR proteins in each frame. (Bottom) Schematic illustration of antisense (C₄G₂)108RO repeats with stop codons inserted in every 12 repeats to prevent the translation of DPR proteins from all reading frames. (B) Immunoblotting of DPR proteins in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75, or 2 repeats. DPR protein levels were detected using anti-FLAG (frame with GP), anti-MYC (frame with PR), and anti-HA (frame with PA). GAPDH was used as a loading control. (C) mRNA levels were measured by quantitative qPCR in cell expressing in_(C₄G₂)75, (C₄G₂)75, or 2 repeats. Error bars represent SD (n = 3). (D, E) Immunoblotting of p-PKR and p-eIF2α in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75, (C₄G₂)108RO, or 2 repeats. p-PKR (T446) and p-eIF2α (Ser51) were normalized to total PKR and eIF2α, respectively. GAPDH was used as a loading control. Error bars represent SD (n = 3 independent experiments). Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. (F, G) Representative images (F) and quantitation (G) of p-eIF2α in primary neurons expressing antisense (C₄G₂)108RO or 2 repeats in the presence and absence of PKR siRNA. Scale bars, 10 µm.

Figure 3—figure supplement 1. Antisense DPR proteins do not activate PKR/eIF2α-dependent integrated stress response.

(A) Representative images and (B) quantification of p-eIF2α staining in HEK293T cells expressing in_(C₄G₂)75, PR50, PA50, or GP80. Green, GP, PA, or PR; red, p-eIF2α; blue, DAPI. Error bars represent SD (n = 2 independent experiments). (C) Representative images of antisense RNA foci in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75 or (C₄G₂)108RO repeats. Foci were detected by RNA FISH. Red, foci; blue, DAPI. (D) Immunoblotting of DPR proteins in HEK293T cells expressing in_(C₄G₂)75, (C₄G₂)75 and (C₄G₂)108RO repeats. DPR protein levels were detected using anti-PR and anti-GP antibodies. GAPDH was used as a loading control. (E) Representative images of antisense RNA foci in primary cortical neurons expressing (C₄G₂)108 detected by RNA FISH. Red, foci; blue, DAPI; Green, MAP2. (F) Immunoblotting and (G) quantification of PKR in primary neurons expressing (C₄G₂)2 and (C₄G₂)108RO together with control or PKR siRNA. PKR were normalized to GADPH. Error bars represent SD. (H) The levels of PKR and p-eIF2α (Ser51) in HEK293T cells expressing (C₄G₂)108RO together with control or PKR siRNA. PKR and p-eIF2α (Ser51) were normalized to GADPH and total eIF2α, respectively. Error bars represent SD (n = 2 independent experiments). Statistical analyses were performed using Student’s t-test. (I) Representative images and quantification of puromycin staining in HEK293T cells expressing (C₄G₂)108RO together with control or PKR siRNA. Error bars represent SD (n = 3 independent experiments). mApple was co-transfected to identify cells with (C₄G₂)108RO expression. Statistical analyses were performed using Student’s t-test. Scale bars, 20 µm (HEK293T), 10 µm (neurons).