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

人IGF2BP蛋白(Insulin like Growth Factor2，简称IMP)是一个由三个高度保守的mRNA结合蛋白组成的家族，分别是IMP1、IMP2和IMP3，具有两个N端RRM基序和四个C端KH结构域的独特模块结构。这里我们关注IMP3，它作为胰腺癌标记物特别有趣。在第一个资助期，我们建立了一个综合的系统战略，将单一结构域解析的SELEX-SEQ，包括基序间隔分析，与体内的iCLIP和结构研究相结合。我们最初的iCLIP分析表明，IMP3主要与mRNAs结合在其3‘-UTR区域，但也在某些内部外显子内。有趣的是，IMP3靶标强烈富含编码膜和分泌蛋白的mRNAs，这表明IMP3在内质网近端定位中具有新的转录后作用。我们的整合方法确定了IMP3的RNA结合特异性和RNP拓扑，涉及所有六个RNA结合域(RBD)和多达五个不同的、间距适当的富含CA和GGC核心RNA元件的簇，覆盖一个100个核苷酸长的目标RNA区域。因此，我们可以解释IMP3-RNA识别的特异性和灵活性，为在基因调控中与多结构域RNA结合蛋白的多价相互作用提供了一个范例(Schneider等人，2018)。同时，我们研究了IMP3与环状RNA(CircRNAs)的特异和稳定的结合，CircRNAs是由前mRNAs通过选择性剪接产生的一类新的非编码RNA。基于对IMP3-免疫共沉淀RNA的RNA-Seq分析，我们确定了含有IMP3的CircRNPs的一个亚组，并对其进行了验证和表征(Schneider等人，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成分的功能谱，重点围绕这三个特定的目标和主题：1.定义和验证通用的IMP3 RNA识别编码；2.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。发现潜在的IMP3介导的mRNA-CircRNA网络。总而言之，我们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。这些研究将依赖于我们在过去三年中建立的IMP3的组合RNA识别代码，我们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生物发生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.