Untangling the contribution of untranslated regions to mRNA translation in health and disease

Abstract: Protein homeostasis is essential for living organisms and a consequence of steps of the gene expression pathway such as transcription, mRNA translation as well as the degradation of mRNA and proteins. These major cellular events demand tight control, high maintenance, and require a large proportion of cellular resources. mRNA translation is regulated by a plethora of cellular pathways as well as features of the mRNA molecule itself and is implicated in malignancies such as cancer. To decipher the contributions of mRNA elements in shaping the proteome, we developed a computational approach to identify mRNA features helping to understand their role in posttranscriptional gene regulation (paper III). While using mTOR-sensitive translation as a model, we characterized an mRNA subset, which despite its transcriptional regulation, does not lead to altered protein levels. This phenomenon, termed translational offsetting, associated with distinct features in the 3’ untranslated region and mRNA stability. The initiation step of mRNA translation is widely considered as rate-limiting and is greatly influenced by features in the 5’ untranslated region. These characteristics can lead to impaired scanning and initiation and have been attributed to cellular cues such as the mTOR pathway and the integrated stress response. In paper II, we applied nano-cap analysis of gene expression (nanoCAGE) and identified 5’UTR variants containing upstream open reading frames. In combination with the development of a reporter-based high-throughput method we studied these variants in a 5’UTR-centric manner, which led to the discovery of an mRNA subset being stressresistant due to precise transcription start site positioning. In paper IV, we studied the coordination of gene expression upon depletion of the transcription factor ERα, known for its role in hormone-dependent cancers. Post-transcriptional regulation upon ERα depletion is characterized by extensive translational offsetting, which is largely assigned to features in the coding sequence of mRNAs. These mRNAs are enriched for codons requiring U34-modified tRNAs for their translation, while these modifications are regulated by ERα. A large proportion of cancer types are characterized by aberrant tumor suppressor activity such as mutations or dysregulated protein levels of p53. Its reactivation by small molecules presents a promising strategy for cancer treatment. However, the exact mode of action of such compounds remains often elusive. RITA, a small molecule initially discovered for its induction of apoptosis upon p53 reactivation, induces cell death in a predominantly p53-independent manner. We studied RITA in the context of mRNA translation and found its activity is dependent on the phosphorylation of eIF2α, a major regulator of mRNA translation (paper I).

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