RNase L localization studies in human cells and potential applications of RNase L modulation in cancer immunotherapy

Abstract

Ribonuclease L is an important effector of the type I IFN pathway in response to viral RNAs. In response to activation by 2-5As, RNase L cleaves both viral and cellular mRNAs. However, there is increasing interest in the roles of RNase L under homeostatic conditions. RNase L has been shown to cleave specific cellular mRNAs that have functions in proliferation and differentiation, but it is still unclear as to how RNase L is able to specifically target these RNAs. In order to elucidate the mechanism of RNase L substrate targeting, this study focused on determining the mechanism through which RNase L is localized. Deep sequencing, immunoblots, and a new localization assay developed for this study, all reveal a nuclear/cytoskeletal enrichment of both RNase L mRNA and protein. In addition, deep sequencing revealed an association of RNase L with RNAs of membrane-bound proteins. Based on this data, it is evident that studying the mechanism of RNase L mRNA localization can reveal how RNase L protein is localized, which has implications for how RNase L targets is substrates. In order to further study the connection between RNase L mRNA and localization of RNase L protein, two RNase L mutants were designed that study different possible mechanisms of RNase L mRNA spatial control of translation.