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

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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.