Impact of RNA-binding proteins and mRNA structures on alternative mechanisms of translational regulation in inflammation-associated tumorigenesis

肿瘤的形成和发展受到免疫系统的强烈影响。具体地说，异常细胞，即恶性细胞，通常被巨噬细胞等免疫细胞识别和清除。在所谓的癌症免疫编辑过程中，肿瘤获得了逃避免疫监视的特征，甚至破坏了免疫细胞，使其成为支持肿瘤的表型。因此，肿瘤微环境包含炎症、免疫调节以及促进增殖的介质。然而，尽管翻译在肿瘤形成和发展中的重要性已被广泛接受，但对肿瘤细胞在炎性肿瘤微环境中的翻译变化知之甚少。在本项目中，我们的目标是在高通量测序数据的实验生成和生物信息学分析的密切合作下，全面表征肿瘤相关细胞因子白介素1β(IL-1β)对人乳腺癌细胞翻译的影响。根据mRNA特异的翻译调控模式通常依赖于mRNAs中的顺式调控元件与反式作用因子(例如RBPs)的相互作用的概念，我们将通过正交有机相分离(OOPS)测序初步确定IL-1β如何影响mRNAs和蛋白质之间的相互作用。由于翻译调控的替代模式，如依赖内部核糖体进入位点(IRES)的翻译起始，被认为与二级RNA结构有关，我们将进一步确定mRNAs对IL-1β的反应在转录组范围内的二级结构的变化。我们预计，相互作用的限制性商业惯例将改变mRNA的结构，这可能再次允许更多的限制性商业惯例与邻近区域结合，从而确保对相关mRNAs翻译的选择性调控。蛋白质-RNA相互作用组和mRNA结构组数据将独立地或联合地与IL-1β诱导的翻译变化相一致。这些比较分析将允许识别mRNAs，其中IL-1β介导的翻译调节与其5‘非翻译区内mRNA结构和/或蛋白质相互作用的变化有关。这一策略应该丰富携带IL-1β反应的IRESS或其他翻译调节元件的mRNAs，如上游开放阅读框架(UORF)。在对选定候选者的翻译调控假设模式进行实验验证后，将确定确切的相互作用限制性商业惯例，并将测试其调控影响。综上所述，我们将建立新的IL-1β响应的IRESS，并对其二级结构和蛋白质相互作用组进行鉴定，最终确定其确切的调控机制。这将对未来炎症相关肿瘤发生中的翻译调节干预感兴趣。

The formation and progression of tumors is strongly influenced by the immune system. Specifically, aberrant, i.e. malignant, cells are usually recognized and removed by immune cells such as macrophages. During the so-called cancer-immunoediting tumors gain features to escape immunological surveillance and even corrupt immune cells into a tumor-supportive phenotype. Consequently, the tumor microenvironment contains inflammatory, immune-modulatory, as well as proliferation promoting mediators. Yet, while the importance of translation on the formation and progression of tumors is widely accepted, little is known about translational changes in tumor cells in response to an inflammatory tumor microenvironment. In the present project, we aim to comprehensively characterize the influence of the tumor-associated cytokine interleukin-1beta (IL-1beta) on translation in human breast carcinoma cells, in close cooperation between the experimental generation of high-throughput sequencing data and the bioinformatics analyses. Following the notion that mRNA-specific modes of translational regulation commonly rely on the interplay of cis-regulatory elements within the mRNAs with trans-acting factors (e.g. RBPs), we will initially determine how IL-1beta affects the interactions between mRNAs and proteins via orthogonal organic phase separation (OOPS)-sequencing. Since alternative modes of translational regulation, such as internal ribosome entry site (IRES)-dependent translation initiation, are considered to be associated with secondary RNA structures, we will further determine changes in the secondary structure of mRNAs in response to IL-1beta in a transcriptome-wide manner. We anticipate that mRNA structures will be altered by interacting RBPs, which again might allow additional RBPs to bind in adjacent regions, thereby ensuring the selective regulation of translation of relevant mRNAs. The protein-RNA interactome and the mRNA structure-ome data will be aligned with the IL-1beta-induced translational changes both independently as well as in combination. These comparative analyses will allow for the identification of mRNAs, where the IL-1beta-mediated translational regulation correlates with changes in mRNA structures and/or protein interactions within their 5’ untranslated regions. This strategy should enrich for mRNAs bearing IL-1beta-responsive IRESs or other translation-regulatory elements, such as upstream open reading frames (uORFs). After the experimental validation of the putative modes of translational regulation of selected candidates, the exact interacting RBPs will be determined and their regulatory impact will be tested. Taken together, we will establish novel IL-1beta-responsive IRESs, characterize their secondary structure and protein interactome, and lastly determine the exact mechanism of regulation. This will be of interest for future translation-modulating interventions in inflammation-associated tumorigenesis.