Fig. 6
Inhibition of RAN translation by eIFs inhibitors and RNA ASOs support a 5′–3′ scanning-dependent mechanism. (a) Illustration of translation inhibitors used to delineate the recruitment of the ribosome at the CUG start codon: 4EIRCat prevents the interaction between eIF4E (4E) and eIF4G (4G). FL3 inhibits RNA helicase eIF4A (4A). Edeine blocks the codon–anticodon interaction. Cycloheximide (CHX) blocks the translational elongation. (b) Translation was performed in presence of CHX, FL3, 4EIRCat, or Edeine in RRL followed by immunoblot detection of anti-HA (poly-GA) antibody. (c–e) HEK293T cells were transfected with the construct #4 expressing 66 G4C2 repeats and treated with FL3 or DMSO control. (c) Immunoblots using antibodies against poly-GA, poly-GP, poly-GR, and HSP-90 proteins. (d) Levels of poly-GA, poly-GP, and poly-GR after FL3 treatment were quantified and normalized to HSP90 and DMSO-treated cells. Graphs represent mean ± SEM, n = 5. Student’s t-test, *** indicate p < 0.001. (e) Levels of repeat-containing transcripts determined by quantitative RT-PCR and normalized to the Rplp0 transcripts and DMSO treated cells. (f) Schematic representations of construct #4 with sequences of sense (RNA-SO) and antisense (RNA-ASO) RNA oligonucleotides used to inhibit RAN translation. (g–h) Translation of capped (G4C2)66 RNAs (construct #4) was performed in RRL in presence of two concentrations of sense or antisense RNA oligonucleotides. (g) [35S]-methionine RAN translation products were detected by autoradiography. (h) Poly-GA and poly-GR were detected by immunoblot using anti-HA (Poly-GA) and -FLAG (Poly-GR) antibodies, respectively
