Mechanistic insights into RAN translation within the Huntingtin coding sequence

Nucleotide repeat expansions support the production of toxic peptides from putatively non-coding regions of the human genome in the absence of an AUG initiation codon. This process, known as repeat-associated non-AUG (RAN) translation, was originally described at CAG repeat expansions with products generated in all three potential reading frames from plasmid-based reporters. Recently, cryptic mis-splicing of mRNAs where the CAG repeat serves as a splice acceptor was proposed as an alternative mechanism by which RAN proteins might be generated. To better understand this process, we generated a series of DNA and RNA based reporters to assess RAN translation in the context of a CAG repeat expansion in exon 1 of the Huntingtin gene (HTT) associated with Huntington Disease. HTT CAG repeats support RAN translation in both the alanine (GCA) and glutamine (CAG) frames in both the presence and absence of upstream AUG start codons or near-AUG cognate codons. HTT RAN translation in all reading frames is less efficient than that observed at CGG or GGGGCC repeats, but it exhibits similar cap-dependence and selective enhancement by activation of the integrated stress response. Importantly, CAG RAN translation was readily detectable from in vitro transcribed RNAs transfected into human cells, rodent neurons or in cell lysates, suggesting that plasmid based aberrant splicing into CAG repeats does not fully explain the observed phenomena. Together, these data suggest that RAN translation from HTT CAG repeats shares key mechanistic parameters with other disease-associated repeat expansions and surrounding RNA contexts.