Investigating Mechanisms of Sequence Specificity and Translation Block in RNase L-mediated Innate Immune Response

Abstract

RNase L is an endoribonuclease vital to the innate immune interferon response to viral ssRNA, and secondarily linked to metabolic conditions and genetic prostate cancer. After activation, RNase L cleaves cellular and viral RNA, causing translational arrest. Although previous research identified its specificity for cleaving at UN^N sites and ability to induce translation block, mechanisms of cleavage and translational arrest have yet to be elucidated. Crystal structure analysis identifies F647 in RNase L as directly interacting with uracil base in RNA. This study aims to better understand the mechanisms behind these two functions through analysis of characterization of F647 mutant variations of RNase L. F647 mutants displayed lower cleavage efficiency than WT and were unable to induce translation block, implying F647's importance to immediate and downstream RNase L function. In addition, F647 mutants did cleave tRNA, suggesting that tRNA fragment presence is not necessary for RNase L-mediated translation block. These findings contrast with the existing polysome traffic jam and signaling models and further support that translation arrest is not due to cleavage of tRNA.