Fig. 2

G₄C₂ RAN translation is cap-dependent and initiates with a methionine. (a) Schemes of the RNA with (G₄C₂)₃₀ (#3) or (G₄C₂)₆₆ (#4) repeats that were transcribed in vitro with T7 RNA polymerase, capped and subjected to translation in RRL. (b) Translation was performed in the presence of [³⁵S]-methionine and capped RNA #3 or #4 at 100 and 200 nM. RAN translation products were detected by autoradiography. Asterisk indicates bands in the stacking gel. (c) Translation was performed in presence of [³⁵S]-methionine and capped RNA #4 followed by immunoprecipitation with antibody against HA-tag and detection of immunoprecipitated [³⁵S]-methionine proteins by autoradiography. (d) Scheme of the canonical translation involving the cap-binding protein eukaryotic initiation factor 4E (eIF4E), the protein platform (eIF4G) and the helicase (eIF4A) that recruit the 40S ribosomal subunit. This pre-initiation complex scans the 5′ of the transcript for an appropriate start codon. Compounds used for the competition assay in (e) and (f) are represented by dark circles and squares for the cap analog (m⁷GpppG) and the inactive form (ApppG), respectively. (e–f) Translation was performed in presence of [³⁵S]-methionine, capped (G₄C₂)₆₆ RNA #4 and an increased concentration of inactive cap (control, ApppG) or cap analog (competitor of the cap, m⁷GpppG). [³⁵S]-methionine RAN translation products and poly-GA were detected by (e) autoradiography and (f) immunoblot with an antibody against HA-tag, respectively