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

RNA结合蛋白的基因突变、缺失或过表达导致转录后调控网络的失控而导致疾病，但其靶向RNA及其调控模式在很大程度上尚不清楚。许多神经退行性疾病是由RBP表达改变引起的，它扰乱了转录后调控网络[1]。例子包括脆性X综合征(FXS)[2]和肌萎缩侧索硬化症(ALS)[3]。本研究旨在了解mRNA靶标的识别，以推导出两个与疾病相关的限制性商业惯例的调控原理，其中一个(FMRP)与FXS和特定类型的自闭症有关，而另一个(TDP-43)的突变与ALS有关。这些限制性商业惯例的分子RNA靶标已经通过PAR-CLIP(光活化核苷酸交联和免疫沉淀)结合高通量测序在宿主组中被鉴定。这种方法可以有效地识别细胞RNA靶标，并从它们的共同序列中确定RNA识别元件。将使用生物物理和结构生物学方法，在存在和不存在与其生物相关的RNA目标以及进一步相互作用的伙伴的情况下分析限制性商业惯例。这种方法将超越对具有分离的RNA结合域的短RNA片段的分析。在存在和不存在配体的情况下，关于限制性商业惯例结构和折叠状态的结果将允许产生关于其分子功能和调节的假说，这将使用体外模型和活细胞进行研究。在分子水平和细胞环境中进一步了解限制性商业惯例可能有助于开发针对涉及限制性商业惯例的疾病的特定药理学干预措施。

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.