Functional characterisation of the non-coding RNA Pantr1 in FOXG1-dependent forebrain development and Rett-syndrome

FOXG1 依赖性前脑发育和 Rett 综合征中非编码 RNA Pantr1 的功能特征

• 批准号: 255240006
• 负责人: Professor Dr. Rolf Backofen
• 金额: --
• 依托单位: Anatomie II: Abteilung für Molekulare Embryologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/255240006?language=en
• 关键词: Functional; characterisation; non; coding; RNA

Rett-syndrome is an autism-spectrum disorder that is characterized by impaired neurodevelopment with e.g. progressive loss of cognitive capabilities, spastic paralysis, ataxia and epilepsy. 90% of Rett-syndrome cases are ascribed to mutations of MECP2, while the rest is due to mutations in other genes such as FOXG1. FOXG1 haploinsufficiency results in atypical Rett syndrome with similar phenotypes. Recent data suggest that both FOXG1 and MECP2 influence expression of long non-coding RNAs (ncRNA) as well as microRNAs and that these ncRNAs are implicated in disease development and progression. Specifically, our preliminary data showed that miR200 family and the long ncRNA (lncRNA) Pantr1 have decreased transcriptional levels in the hippocampus of Foxg1 heterozygote adult mice. To carry on our initial findings on the role of ncRNAs in atypical Rett-syndrome, we aim to analyse molecular functions of Pantr1 within this project. Bridging the fields of bioinformatics, molecular biology, and biochemistry our approaches to elucidate the function of Pantr1 in neural cells include target prediction and validation, as well as prediction and validation of transcriptional and post-transcriptional regulatory mechanisms. Specifically, we will explore the function of the Pantr1-PURb lncRNA-protein complex that we identified in our preliminary work. Within the proposed project we will (1) determine alterations of the transcriptome after changing expression levels of Pantr1 and PURb using RNA-seq, (2) use chromatin immunoprecipitation by RNA pull down (ChIRP) to analyse whether PURb promotes or hampers Pantr1 from binding to DNA, (3) employ Pantr1 ChIRP followed by mass spectrometry in conditions with and without PURb expression to elucidate whether PURb-binding interferes with association of Pantr1 to other RNA binding proteins, and (4) use iCLIP and ChIP to examine whether binding to Pantr1 interferes with binding of PURb to RNA or DNA. Further, we intend to rescue Pantr1 and miR200 family expression in vivo to analyse if this can revert phenotypical alterations in the adult mouse hippocampus. In addition, we will extend our results to FOXG1-syndrome patient-specific iPSC-derived neurons by studying Pantr1 and miR200 family expression and molecular function. Together, these data on the role of ncRNAs in atypical Rett syndrome will significantly further our understanding of the molecular basis of this neurodevelopmental disorder and may open avenues for novel therapeutic strategies.

雷特综合征是一种自闭症谱系障碍，其特征在于神经发育受损，例如认知能力的进行性丧失、痉挛性麻痹、共济失调和癫痫。90%的雷特综合征病例归因于MECP 2突变，而其余的则是由于其他基因如FOXG 1突变。FOXG 1单倍不足导致具有相似表型的非典型Rett综合征。最近的数据表明，FOXG 1和MECP 2都影响长链非编码RNA（ncRNA）和microRNA的表达，这些ncRNA与疾病的发展和进展有关。具体来说，我们的初步数据显示，miR 200家族和长ncRNA（lncRNA）Pantr 1在Foxg 1杂合子成年小鼠海马中的转录水平降低。为了继续我们对ncRNA在非典型Rett综合征中作用的初步发现，我们的目标是在本项目中分析Pantr 1的分子功能。桥接生物信息学，分子生物学和生物化学领域，我们的方法来阐明Pantr 1在神经细胞中的功能，包括目标预测和验证，以及预测和验证的转录和转录后调控机制。具体来说，我们将探索我们在前期工作中鉴定的Pantr 1-PURb lncRNA-蛋白质复合物的功能。在拟议的项目中，我们将（1）使用RNA-seq确定改变Pantr 1和PURb表达水平后转录组的变化，（2）使用RNA下拉染色质免疫沉淀（ChIRP）分析PURb是否促进或阻碍Pantr 1与DNA结合，（3）在有和没有PURb表达的条件下，采用Pantr 1 ChIRP，然后进行质谱分析，以阐明PURb-结合干扰Pantr 1与其他RNA结合蛋白的结合，和（4）使用iCLIP和ChIP来检查与Pantr 1的结合是否干扰PURb与RNA或DNA的结合。此外，我们打算拯救Pantr 1和miR 200家族在体内的表达，以分析这是否可以恢复成年小鼠海马的表型改变。此外，我们将通过研究Pantr 1和miR 200家族的表达和分子功能，将我们的结果扩展到FOXG 1综合征患者特异性iPSC衍生的神经元。总之，这些关于ncRNA在非典型Rett综合征中作用的数据将显着促进我们对这种神经发育障碍的分子基础的理解，并可能为新的治疗策略开辟途径。