Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...

Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap-abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt. Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. Gene mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt. Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden. / Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F protein complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E‑BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. Genes with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap-independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project.