Validation of select novel IRP target mRNAs with in vitro transcripts. In vitro transcribed full-length mRNAs were incubated at a 40× molar excess against an H-ferritin IRE-radiolabeled probe for the binding of recombinant IRP1. Wt corresponds to an ∼ 1-kb reporter mRNA bearing the 5′ IRE of H ferritin mRNA; mut is the same reporter with a deltaC14 mutation of the IRE. Black bars represent negative control mRNAs (Renilla) or no competitor signal. Yellow bars represent positive controls (H-ferritin reporters and full-length Fth1 and Slc40a1 mRNAs). Red and blue bars correspond to novel IRP target mRNAs with or without a bioinformatically predicted IRE-like motif, respectively. Tested full-length and indicated 5′ restriction enzyme truncation mRNAs are grouped together. Above each group a schematic representation is shown indicating the restriction enzyme used to assess truncated forms and the location of the predicted IRE motif (round hairpin) or the putative IRP-binding RNA region (squared hairpin). P values are reported (***P < .001, **P < .01, *P < .05, unpaired 2-tailed Student t test compared with the mutant H-ferritin IRE construct, mut, or with each corresponding non-IRE construct). Data are presented as mean ± SEM.