|dc.description.abstract||RNA-binding proteins (RBPs) are involved in many cellular processes and thereby contribute to the regulation of gene expression. As a consequence, mutation or altered expression of RBPs can cause diseases such as cancer. Members of the insulin-like growth factor 2 mRNA binding protein (IMP, IGF2BP) family are recognised tumour markers as they are upregulated or de novo expressed in a variety of cancer types. They are known to regulate RNAs in terms of stability, translation and localisation. However, the overall picture of their biological function is still unclear. Developing strategies to inhibit these proteins could thus promote new anti-cancer therapies, but requires also a better understanding of the underlying biological functions.
This thesis focusses on the tumour marker and multidomain RBP IMP3. In a first step, IMP3 was targeted by designer circRNAs, which should functionally inhibit IMP3 by competing with endogenous RNA for its binding. Initial in vitro experiments revealed high binding affinities for IMP3 to circRNA sponges. To examine if this holds also true in vivo, two different circRNA expression systems were tested: circRNAs were either stably integrated into the genome and could be inducibly expressed, or circRNA expression was plasmid-driven and mediated by the so-called Tornado ribozyme system. Interactions of IMP3 with circRNAs were then captured in vivo by RNA-immunoprecipitation assays. This way, binding of our designer circRNAs could be confirmed in cell culture and represents a new strategy for inhibiting RBPs in the context of anti-cancer therapies.
Concerning the biological function of IMP3, we had preliminary evidence that it might operate in the secretory pathway. Thereby, mRNAs are guided to the endoplasmic reticulum (ER) for local translation, and the resulting protein is translocated into the ER lumen. To obtain more insight on a global transcriptome level, subcellular fractionation was combined with next-generation RNA-sequencing (RNA-seq) and individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) in human ES-2 cells. We focussed on mRNAs with IMP3-dependent changes of gene expression levels in the membrane organelle fraction. A subset of direct mRNA targets was identified to be downregulated upon IMP3 knockdown. Biochemical validation by IMP3 RIP experiments and RT-qPCR of subcellular extracts confirmed our results and revealed certain mRNAs of the secretory pathway to be additionally regulated by IMP3. In a last step, the effect of our designer circRNA sponges on target mRNA localisation was tested. In sum, our results support a model in which IMP3 interactions with the mRNA 3’-UTR provide an additional localisation signal to direct specific mRNAs into the secretory pathway.||de_DE