The multidomain RNA-binding protein IMP3:combinatorial RNA recognition and functions during mRNP biogenesis

人IGF 2BP蛋白（胰岛素样生长因子2 mRNA结合蛋白;或简称IMP）是三种高度保守的mRNA结合蛋白IMP 1、IMP 2和IMP 3的家族，其特征在于具有两个N-末端RRM基序和四个C-末端KH结构域的独特模块结构。在这里，我们关注IMP 3，它作为胰腺癌标志物特别有趣。在第一个资助期内，我们建立了一个综合的系统策略，将单域解析的SELEX-seq（包括基序间距分析）与体内iCLIP和结构研究相结合。我们最初的iCLIP分析表明，IMP3主要在其3 '-UTR区域内结合mRNA，但也在某些内部外显子内结合。有趣的是，IMP3的目标是强烈富集的mRNA编码的膜和分泌蛋白，这表明一个新的，转录后的IMP3在ER近端定位的作用。我们的综合方法鉴定了IMP 3的RNA结合特异性和RNP拓扑结构，涉及所有六个RNA结合结构域（RBD）和一簇多达五个不同的且适当间隔的富含CA和GGC的核心RNA元件，覆盖>100个核苷酸长的靶RNA区域。因此，可以解释IMP 3-RNA识别的特异性和灵活性，为基因调控中与多结构域RNA结合蛋白的多价相互作用的功能提供范例（Schneider et al.，与此同时，我们研究了IMP 3与环状RNA（circRNA）的特异性和稳定性相关性，circRNA是一类通过选择性剪接由前mRNA产生的新的非编码RNA。基于IMP 3-共免疫沉淀的RNA的RNA-Seq分析，我们鉴定了一个亚组的含有IMP 3的circRNP，其被验证和表征（Schneider et al.，2016）。 在此基础上，我们计划更详细地研究RNA识别的分子机制和IMP3作为mRNP组分的功能谱，重点关注以下三个具体目标和主题：1.定义并验证IMP 3的通用RNA识别码。寻找IMP3蛋白在mRNA生命周期中的功能作用：3'加工，稳定性，细胞定位，抑制3。揭示潜在的IMP3介导的mRNA-circRNA网络。 总之，我们的项目在第二个资助期的主要目标将是研究IMP3的功能谱，IMP3是一种典型的多结构域RNA结合蛋白。这些研究将依赖于IMP3的组合RNA识别代码，我们在过去三年中建立了该代码，并将继续对其进行进一步评估。同时，我们将继续寻找IMP3介导的mRNP和circRNP生物合成途径之间的功能联系和调控网络。

The human IGF2BP proteins (Insulin like Growth Factor 2 mRNA Binding-Protein; or shortly IMP) are a family of three highly conserved mRNA-binding proteins, IMP1, IMP2, and IMP3, characterized by a unique modular structure with two N-terminal RRM motifs and four C-terminal KH domains. Here we focus on IMP3, which is particularly interesting as a pancreatic cancer marker. In the first funding period we established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, including motif-spacing analyses, with in vivo iCLIP and structural studies. Our initial iCLIP analyses had shown that IMP3 binds mRNAs primarily within their 3’-UTR regions, but also within certain internal exons. Interestingly, IMP3 targets are strongly enriched for mRNAs coding for membrane and secretory proteins, suggesting a new, post-transcriptional role of IMP3 in ER-proximal localization. Our integrative approach identified the RNA-binding specificity and RNP topology of IMP3, involving all six RNA-binding domains (RBDs) and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Thereby both specificity and flexibility of IMP3-RNA recognition can be explained, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation (Schneider et al., 2018).In parallel we investigated the specific and stable association of IMP3 with circular RNAs (circRNAs), a new class of noncoding RNAs generated from pre-mRNAs by alternative splicing. Based on RNA-Seq analysis of IMP3-coimmunoprecipitated RNA, we identified a subgroup of IMP3-containing circRNPs, which were validated and characterized (Schneider et al., 2016). On this basis, we plan to investigate in more detail the molecular mechanisms of RNA recognition and the functional spectrum of IMP3 as an mRNP component, focussing on these three specific aims and topics: 1. Defining and validating the general IMP3 RNA-recognition code,2. Searching for functional roles of IMP3 protein in the mRNA life cycle: 3’ processing, stability, cellular localization, translation3. Uncovering potential IMP3-mediated mRNA-circRNA networks. In sum, the major aim of our project in the second funding period will be to investigate the functional spectrum of IMP3, a prototypical multidomain RNA-binding protein. These studies will rely on the combinatorial RNA-recognition code for IMP3, which we established in the last three years and which we continue to evaluate further. In parallel, we will pursue our search for IMP3-mediated functional links and regulatory networks between mRNP and circRNP biogenesis pathways.