The impact of the RNA-binding protein hnRNP-A1 on translation in inflammation-associated tumorigenesis

炎性肿瘤微环境是由浸润的免疫细胞形成的，尤其是巨噬细胞，巨噬细胞释放促进恶性转化的促炎介质。因此，炎症条件被广泛接受支持肿瘤发生的过程。我们之前已经表明，巨噬细胞相关的炎症条件，除了诱导转录变化外，还会引起肿瘤细胞的翻译变化，从而促进肿瘤的发展。初步数据表明，在这些条件下，hnRNP-A1可能在调节帽非依赖性翻译变化中发挥重要作用。因此，我们现在的目标是识别和表征肿瘤细胞中依赖rna结合蛋白hnRNP-A1的炎症诱导的翻译变化。使用多体分离，我们将评估炎症条件下肿瘤细胞中hnrnp - a1依赖的翻译变化。此外，我们将通过PAR-CLIP（光激活核糖核苷增强交联和免疫沉淀）分析确定hnRNP-A1与其mRNA靶标的确切结合位点。结合这两种方法，我们将确定通过5'非翻译区与hnRNP-A1相互作用的翻译调节的hnRNP-A1靶点。这些靶标将被分析是否存在内部核糖体进入位点（IRES）——促进帽独立翻译起始的元件。确定共享序列或结构基序可以预测hnRNP-A1反应性IRESs。进一步的分析旨在确定潜在的co-IRES交易因子（ITAFs），这些因子有助于选定hnRNP-A1靶点的ires依赖翻译。最后，巨噬细胞依赖性炎症因子对ires活性改变的影响及其信号级联的阐明将为翻译靶向干预提供基础。

The inflammatory tumor microenvironment is shaped by infiltrating immune cells, most notably macrophages, which release pro-inflammatory mediators that contribute to malignant transformation. In line, inflammatory conditions are widely accepted to support the process of tumorigenesis. We have previously shown that macrophage-associated inflammatory conditions, in addition to inducing transcriptional changes, elicit translational changes in tumor cells that foster tumor development. Preliminary data imply that hnRNP-A1 could play an important role in the regulation of cap-independent translation changes under these conditions. Thus, we now aim to identify and characterize inflammation-induced translational changes in tumor cells that depend on the RNA-binding protein hnRNP-A1. Using polysomal fractionation, we will assess hnRNP-A1-dependent translational changes in tumor cells under inflammatory conditions. In addition, we will determine the exact binding sites of hnRNP-A1 on its mRNA targets by PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) analysis. Combining both approaches we will identify translationally regulated hnRNP-A1 targets that interact with hnRNP-A1 via their 5' untranslated region. These targets will be analyzed for the presence of internal ribosome entry site (IRES)-elements facilitating cap-independent initiation of translation. Determination of shared sequences or structural motifs will predict hnRNP-A1 responsive IRESs. Further analyses aim at identifying potential co-IRES trans-acting factors (ITAFs) contributing the IRES-dependent translation of selected hnRNP-A1 targets. Finally, the elucidation of the macrophage-dependent inflammatory factors contributing to the altered IRES-activities and the contributing signaling cascades will provide the basis for translation-targeted interventions.