Mechanistic insights into RAN translation within the Huntingtin coding sequence

Abstract

Nucleotide repeat expansions support production of toxic peptides from putatively non-coding regions of the human genome without use of an AUG initiation codon. This process, known as RAN translation, was originally described at CAG repeats with products generated in all three possible reading frames. Recently, cryptic mis-splicing of mRNAs in which the CAG repeat acts as a splice acceptor was proposed as an alternative mechanism for RAN protein production. To investigate this, we generated 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, with or without upstream AUG start codons or near-AUG cognate codons, and at levels comparable to RAN translation from CGG and GGGGCC repeats. CAG RAN translation products were readily detectable from in vitro transcribed RNAs transfected into human cells and rodent neurons - suggesting that plasmid based aberrant splicing into CAG repeats cannot fully explain the observed phenomena. These findings indicate that RAN translation from HTT CAG repeats shares key mechanistic parameters with other disease-associated repeat expansions. Key PointsO_LITranslation at HTT CAG repeats occurs in at least two reading frames absent any AUG start codon. C_LIO_LILike RAN translation at other repeats, HTT CAG RAN translation is enhanced by cell stress. C_LIO_LIHTT CAG RAN translation readily occurs from in vitro transcribed RNAs in neurons. C_LI