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

人IGF2BP蛋白（胰岛素样生长因子2 mRNA结合蛋白，简称IMP）是一个由三个高度保守的mRNA结合蛋白IMP1、IMP2和IMP3组成的蛋白家族，具有独特的模块化结构，具有两个n端RRM基基和四个c端KH结构域。这里我们关注的是作为胰腺癌标志物的IMP3，它特别有趣。在第一个资助期，我们建立了一个综合的系统策略，结合单域解析SELEX-seq，包括基序间距分析，体内iCLIP和结构研究。我们最初的iCLIP分析表明，IMP3主要结合mrna的3 ' -UTR区域，但也结合某些内部外显子。有趣的是，IMP3靶点富含编码膜和分泌蛋白的mrna，这表明IMP3在er近端定位中具有新的转录后作用。我们的综合方法确定了IMP3的RNA结合特异性和RNP拓扑结构，涉及所有6个RNA结合域（rbd）和多达5个不同且间隔适当的CA-rich和GGC-core RNA元件，覆盖了bb100个核苷酸长的靶RNA区域。因此，可以解释IMP3-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了范例（Schneider等人，2018）。同时，我们研究了IMP3与环状rna （circRNAs）的特异性和稳定关联，环状rna是一类由前mrna通过选择性剪接产生的新型非编码rna。基于对imp3共免疫沉淀RNA的RNA- seq分析，我们确定了一个含有imp3的circrnp亚组，并对其进行了验证和表征（Schneider et al., 2016）。在此基础上，我们计划更详细地研究RNA识别的分子机制和作为mRNP成分的IMP3的功能谱，重点研究以下三个具体目标和主题：1。定义和验证通用的IMP3 rna识别代码；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、翻译3。揭示潜在的imp3介导的mRNA-circRNA网络。总之，我们项目在第二个资助期的主要目标是研究IMP3的功能谱，这是一种典型的多结构域rna结合蛋白。这些研究将依赖于我们在过去三年中建立的IMP3组合rna识别代码，我们将继续进一步评估。同时，我们将继续寻找mRNP和circRNP生物发生途径之间的imp3介导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.