Some mRNAs with potential alternative translation initiation sites (TISs) may produce in-frame alternative protein isoforms (proteoforms) that have extended or truncated N termini. Only a few N-terminal proteoforms have been characterized and proteoform regulation and function remain unclear. Lee, Sun et al. now show that NPTXR mRNA, which encodes the neuronal pentraxin receptor (NPR), has TISs that yield proteoforms with distinct localization and functions.

Both of these TISs are conserved in vertebrates. In vitro translation of NPR reporter mRNAs from human, mouse, frog or zebrafish showed that alternative use of NPR TISs is evolutionarily conserved, but CUG initiation is substantially higher in humans and mice. In silico analysis predicted the formation of a stable RNA stem–loop structure, 10–12 nucleotides downstream of the CUG TIS in humans and mice; mutation and disruption of this structure substantially reduced the efficiency of CUG initiation, which was restored by introducing compensatory mutations. Thus, the mRNA secondary structure is required and sufficient for CUG TIS use in mammals.