Viral infections remain a challenging health issue worldwide and, in many cases, treatment options are limited. Many viruses, such as Influenza A and Hepatitis C, trigger the RNase L pathway in human cells. RNase L is activated by a cascade of events: the release of interferons upon virus recognition, activation of the JAK/Stat signaling pathway, transcription of oligoadenylate synthetase (OAS) genes and production of 2-5- oligo-adenylate (2-5A). 2-5A promotes dimerization and activation of the endoribonuclease RNase L that cleaves the single stranded regions of viral and host mRNAs. Cleavage of these RNAs leads to physiological changes in the cell, such as apoptosis and decreased cell motility. Besides the central role of RNase L in viral infections, mutations in the RNASEL gene are associated with specific cancer types, including hereditary prostate cancer. In addition to the canonical endonuclease role of RNase L, it has been suggested that this enzyme plays a role in translation termination failure and allows ribosomes to access the 3' untranslated region (3'UTR) downstream of the stop codon. Therefore, in specific aim 1, I will investigate the occurrence of ribosomes in the 3'UTR region of mRNAs at the global and local (individual gene) level by ribosome footprint profiling in RNase L activated human cells. RNase L is known to directly interact with the ribosome termination/recycling factors ABCE1 (ATP-binding cassette E1) and eRF3 (eukaryotic release factor 3). However, it is unknown if other factors or the ribosome itself interacts with RNase L. Therefore, in my first aim, I will also investigate and map the potential interaction partners of RNase L by immunoprecipitation, mass-spectrometry and recombinant protein binding assays. In addition, I will perform analysis of 3'UTR ribosome occupancy in recycling factor depleted cells to study the mechanism by which RNase L modulates the presence of ribosomes in the 3'UTR. It has been suggested that ribosomes that access the 3'UTR continue translating in RNase L activated cells. To investigate this possibility, I will employ a novel technique, real-time fluorescent single molecule detection of translating nascent peptides (SINAPS), in living cells in combination with western blotting in specific aim 2. Furthermore, the potential functions of these 3'UTR translated peptides are unknown. It has been suggested that 3'UTR translation may produce short peptides that can be loaded onto MHC-I (Major Histocompatibility Complex Class I) and presented on the surface of cells, triggering further immune response. To directly test this hypothesis in aim 2 I will detect specific 3'UTR translated peptides on MHC-I molecules. In summary, the proposed project will investigate unexplored outcomes of RNase L activation. Uncovering new aspects of the defense against viral infections will enable further studies and potentially contribute to the development of new therapeutic strategies.
The persistent presence and continuing emergence of viral infections is one of the major obstacles to improved human health. The majority of viruses, such as human immunodeficiency virus 1, activate an endoribonuclease, RNase L, that is proposed to induce translation termination failure and non-canonical 3'UTR translation by the ribosome. Therefore, in the present proposal I will investigate the potential mechanism and function of this unexplored feature of RNase L activation with the combination of new innovative and classical molecular biology techniques.