Structural studies on mRNA recognition by FMRP and TDP-43, two RNA-binding proteins associated with neuronal diseases

通过基因突变、RNA结合蛋白的缺失或过表达导致的转录后调控网络的失调导致疾病，然而它们的靶RNA及其调控模式在很大程度上是未知的。许多神经退行性疾病是由改变的RBP表达引起的，其扰乱了转录后调控网络[1]。例如脆性X综合征（FXS）[2]和肌萎缩侧索硬化症（ALS）[3]。该研究项目旨在了解mRNA靶点的识别，以推断两种疾病相关RBP的调控原则，其中一种（FMRP）与FXS和特定类型的自闭症相关，而另一种（TDP-43）的突变与ALS相关。通过使用PAR-CLIP（光活化核糖核苷酸交联和免疫沉淀）结合高通量测序，在宿主组中鉴定了这些RBP的分子RNA靶标。该方法允许有效地鉴定细胞RNA靶标并从其共有序列中确定RNA识别元件。将使用生物物理学和结构生物学方法，在存在和不存在其生物学相关RNA靶标以及进一步相互作用伴侣的情况下分析RBP。这种方法将超越与分离的RNA结合域的短RNA片段的分析。在存在和不存在其配体的情况下的RBPs的结构和折叠状态的结果将允许产生其分子功能和调节的假设，这将使用体外模型和活细胞进行研究。进一步了解RBPs在分子水平以及在细胞环境中可能有助于开发特定的药物干预涉及疾病的RBPs。

The deregulation of posttranscriptional regulatory networks by genetic mutation, deletion or overexpression of RNA-binding proteins results in disease, yet their target RNAs and their modes of regulation are largely unknown. Many neurodegenerative conditions are caused by altered RBP expression that perturbs post-transcriptional regulatory networks [1]. Examples include Fragile-X-syndrome (FXS) [2] and amyotrophic lateral sclerosis (ALS) [3]. This research project aims to understand the recognition of mRNA targets to deduce the regulatory principles of two disease-associated RBPs, one of which (FMRP) is associated with the FXS and particular types of autism, whereas mutations in the other (TDP-43) are associated with ALS. Molecular RNA targets of these RBPs have been identified in the host's group by using PAR-CLIP (photoactivatable ribonucleotide crosslinking and immunoprecipitation) in combination with high-throughput sequencing. This method allows an efficient identification of cellular RNA targets and the determination of RNA recognition elements from their consensus sequences. The RBPs will be analyzed in the presence and absence of their biologically relevant RNA targets as well as further interaction partners using biophysical and structural-biology methods. This approach will go beyond analyses of short RNA fragments with isolated RNA-binding domains. Results on the structure and folding states of the RBPs in the presence and absence of their ligands will allow the generation of hypotheses of their molecular function and regulation, which will be investigated using in vitro models and living cells. A further understanding of RBPs at the molecular level as well as in a cellular environment may contribute to the development of specific pharmacological interventions for RBP-involving diseases.