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

瑞特综合征是一种自闭症谱系障碍，其特征是神经发育受损，例如认知能力进行性丧失、痉挛性麻痹、共济失调和癫痫。90%的rett综合征病例归因于MECP2的突变，而其余病例则归因于其他基因如FOXG1的突变。FOXG1单倍不全导致具有相似表型的非典型Rett综合征。最近的数据表明，FOXG1和MECP2都影响长链非编码rna （ncRNA）和microrna的表达，这些ncRNA与疾病的发生和进展有关。具体而言，我们的初步数据显示，miR200家族和长ncRNA (lncRNA) Pantr1在Foxg1杂合子成年小鼠海马中的转录水平降低。为了继续我们关于ncrna在非典型rett综合征中的作用的初步发现，我们的目标是在这个项目中分析Pantr1的分子功能。结合生物信息学、分子生物学和生物化学领域，我们研究Pantr1在神经细胞中的功能的方法包括靶标预测和验证，以及转录和转录后调控机制的预测和验证。具体来说，我们将探索我们在初步工作中发现的Pantr1-PURb lncrna -蛋白复合物的功能。在该项目中，我们将(1)使用RNA-seq测定Pantr1和PURb表达水平改变后转录组的变化，(2)使用染色质免疫沉淀法RNA拉下（ChIRP）分析PURb是否促进或阻碍Pantr1与DNA的结合，(3)在有和没有PURb表达的条件下，使用Pantr1 ChIRP和质谱法来阐明PURb结合是否干扰Pantr1与其他RNA结合蛋白的结合。(4)利用iCLIP和ChIP检测与Pantr1的结合是否会干扰PURb与RNA或DNA的结合。此外，我们打算在体内恢复Pantr1和miR200家族的表达，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Pantr1和miR200家族表达和分子功能，将我们的研究结果扩展到foxg1综合征患者特异性ipsc衍生神经元。总之，这些关于ncrna在非典型Rett综合征中的作用的数据将大大加深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道路。

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.