Mutations within the hnRNP C or the UC(U)8 site on the unr IRES impair the internal translation initiation of the unr mRNA. (A) Unr IRES RNAs. (B) Translation activation of WT and mutated unr IRES sequences by hnRNP C. The activity of the IRES represented in the bar chart was calculated as the normalized averaged ratio of the Firefly over Renilla luminescence; standard deviations (SD) were estimated from a minimum of four independent replicates. (C) Schematic representation of hnRNP C variants. (RRM) RNA recognition motif; (C2) 13 amino acid insertion in the C2 variant; (UR) unstructured region; (BR) basic region, (OD) oligomerization domain; (CTD) carboxy-terminal domain; (F) Flag tag. Red arrows indicate the position of mutated residues. (D) Unr IRES activation with wild-type and mutated ectopic hnRNP C1 in the absence of endogenous hnRNP C protein. The quantity of soluble hnRNP C in the cellular extracts was assessed by Western blot against the carboxy-terminal epitope 4F4 and/or against the Flag tag. (E) Effect of hnRNP C1 mutants on the IRES activity in the presence of endogenous hnRNP C.
