Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12.
Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12.
复制标题
12 型脊髓小脑共济失调中 PPP2R2B 基因位点的双向转录。
DOI：
10.1101/2023.04.02.535298
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Li,PanP
中科院分区：
文献类型：
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作者：
Zhou,Chengqian;Liu,HansB;Bakhsh,FatemehJ;Wu,Bin;Ying,Mingyao;Margolis,RussellL;Li,PanP
背景脊髓小脑性共济失调12型（Spinocerebellar ataxia type 12，SCA 12）是一种由PPP 2 R2 B基因CAG重复序列扩增引起的神经退行性疾病。方法在SCA 12人诱导多能干细胞（iPSCs）中检测PPP 2 R2 B-AS 1转录本的表达，iPSC衍生的NGN 2神经元和SCA 12敲入小鼠脑，使用链特异性逆转录聚合酶链反应。在SCA 12细胞模型中，通过荧光原位杂交检测了扩增PPP 2 R2 B-AS 1（expPPP 2 R2 B-AS 1）RNA形成病灶的趋势，这是涉及突变RNA的毒性过程的标志。通过caspase 3/7活性检测expPPP 2 R2 B-AS 1转录物对SK-N-MC神经母细胞瘤细胞的凋亡作用。Western blot检测SK-N-MC细胞中PPP 2 R2 B-AS 1转录本的重复序列相关非ATG启动翻译的表达。结果PPP 2 R2 B基因位点的重复序列在SCA 12 iPSC、iPSC衍生的NGN 2神经元和SCA 12小鼠脑中双向转录。转染的pPPP 2 R2 B-AS 1转录物诱导SK-N-MC细胞凋亡，并且凋亡效应可能至少部分由RNA二级结构介导。expPPP 2 R2 B-AS 1转录本在SK-N-MC细胞中形成CUG RNA灶。expPPP 2 R2 B-AS 1转录本通过重复相关非ATG翻译在丙氨酸开放阅读框（ORF）中翻译，CUG重复内的单核苷酸中断和MBNL 1过表达会减少这种翻译。结论这些发现表明PPP 2 R2 B-AS 1参与SCA 12的发病机制，因此可能为该疾病提供新的治疗靶点。© 2023 International Parkinson and Movement Disorder Society。
BackgroundSpinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in thePPP2R2Bgene.ObjectiveIn this study, 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 reverse transcription polymerase chain reaction. 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 fluorescence in situ hybridization. The apoptotic 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 translation ofexpPPP2R2B‐AS1transcript in SK‐N‐MC cells.ResultsThe repeat region in thePPP2R2Bgene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC‐derived NGN2 neurons, and SCA12 mouse brains. TransfectedexpPPP2R2B‐AS1transcripts induce apoptosis in SK‐N‐MC cells, and the apoptotic effect 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 open reading frame (ORF) via repeat‐associated non‐ATG translation, which is diminished by single‐nucleotide interruptions within the CUG repeat and MBNL1 overexpression.ConclusionsThese findings suggest thatPPP2R2B‐AS1contributes to SCA12 pathogenesis and may therefore provide a novel therapeutic target for the disease. © 2023 International Parkinson and Movement Disorder Society.