Bidirectional transcription at thePPP2R2Bgene locus in spinocerebellar ataxia type 12

Abstract

AbstractOBJECTIVESpinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in thePPP2R2B gene. Here we tested the hypothesis that thePPP2R2B antisense(PPP2R2B-AS1) transcript containing a CUG repeat is expressed and contributes to SCA12 pathogenesis.METHODSExpression ofPPP2R2B-AS1transcript was detected in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific RT-PCR (SS-RT-PCR). The tendency of expandedPPP2R2B-AS1(expPPP2R2B-AS1) RNA to form foci, a marker of toxic processes involving mutant RNAs, was examined in SCA12 cell models by fluorescencein situhybridization. The toxic effect ofexpPPP2R2B-AS1transcripts on SK-N-MC neuroblastoma cells was evaluated by caspase 3/7 activity. Western blot was used to examine the expression of repeat associated non-ATG-initiated (RAN) translation ofexpPPP2R2B-AS1transcript in SK-N-MC cells.RESULTSThe repeat region inPPP2R2Bgene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 mouse brains. TransfectedexpPPP2R2B-AS1transcripts are toxic to SK-N-MC cells, and the toxicity may be mediated, at least in part, by the RNA secondary structure. TheexpPPP2R2B-AS1transcripts form CUG RNA foci in SK-N-MC cells.expPPP2R2B-AS1transcript is translated in the Alanine ORF via repeat-associated non-ATG (RAN) translation, which is diminished by single nucleotide interruptions within the CUG repeat, and MBNL1 overexpression.INTERPRETATIONThese findings suggest thatPPP2R2B-AS1contributes to SCA12 pathogenesis, and may therefore provide a novel therapeutic target for the disease.