The role of host mRNA cleavage by RNase L in viral infections - PROJECT SUMMARY Viral infections remain a challenging public health issue worldwide. The presence of many viruses, such as Influenza A and Hepatitis C, activate a latent Ribonuclease (RNase L) in human cells. Activated RNase L cleaves the single stranded regions of viral and host mRNAs. Cleavage of these RNAs leads to physiological changes in the cell, such as autophagy, senescence, decreased cell motility, interferon production and cell death. However, many aspects of RNase L activation in cells are not yet understood. Recent in vivo and in vitro kinetic studies suggested that RNA cleavage by RNase L is modulated in the cell by an unidentified factor. RNase L can directly interact with the ribosome and with several translation factors involved in different steps of protein synthesis. Due to these interactions it was proposed that RNase L’s cleavage activity is modulated by the translation of the host messenger RNAs (mRNA). Therefore, in specific aim 1, I will investigate this relationship between host mRNA cleavage by RNase L and translation at the global and individual gene level by combining two high-throughput sequencing methods, ribosome footprint profiling and RNA sequencing in RNase L activated human cells. In addition, ribosome-mediated RNase L cleavage activity will be also directly observed at the individual gene level by a novel technique, the real-time fluorescent single molecule detection of translating nascent peptides (SINAPS) in living cells. In aim 2, I will also explore the potential involvement of translation factors in translation-mediated RNA cleavage by RNase L by first mapping the details of interactions of RNase L and translation factors by mutagenesis studies and Cryo-Electron Microscopy. Then these interactions will be disrupted in living cells to probe their potential involvement in mediating RNA cleavage by RNase L. Furthermore, RNase L activation can lead to many physiological changes in the cell, such as autophagy and apoptosis. It is plausible that the level of active RNase L is the determinant of which physiological processes will occur. To investigate the correlation between active RNase L levels and RNase L mediated changes in the cell, I will develop a fluorescent RNase L activation indicator that will allow us to sort cells into homogenously activated populations in aim 3. Subsequently, in these sorted cells I will detect changes in the transcriptome and translatome and assess signatures of physiological changes of autophagy and apoptosis in the context of RNase L activation levels. In addition, I will also study how RNase L cleaved host RNA fragments contribute to activation of innate immune response pathways in cells by using Cross-linking Co-Immunoprecipitation and sequencing (CLIP-seq). In summary, the proposed project will investigate unexplored aspects and 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.
