Dicer DNA nickase activity and its role in anti-viral immunity in human cells

摘要

Abstract Dicer is a multi-domain ribonuclease III (RNase III) enzyme that produces small interfering RNAs (siRNAs) and microRNAs (miRNAs) from dsRNA precursors. RNA silencing mediated by these small RNAs is an important defense system against viral infection. Loss of Dicer causes loss of anti-viral immunity. Previous study in C. elegans showed that cleavage of the C. elegans RNase Dicer by pro- apoptotic caspase-3 produces a stable C-terminal fragment of Dicer that now functions as a deoxyribonuclease (DNase) in the developmental apoptosis pathway. However, even more than a decade after this study, whether the RNase-to-DNase conversion of Dicer by specific proteolysis occurs in any other animals including human remains unknown. Whether Dicer DNase has any role in anti-viral immunity also remains unknown. When cells are infected by viruses despite their anti-viral system, many of them undergo programmed cell death (PCD or apoptosis), which helps to abort the production and release of progeny viruses from the cells. Growing evidence indicates that some proteins have both ‘day- jobs’ in healthy cells and ‘death-jobs’ in cells undergoing PCD. Thus, evolution may have linked day-jobs and death-jobs in the same molecule to ensure that cell death is appropriately linked to and regulated by multiple normal cellular processes. In this proposal, we hypothesize that human Dicer has day-jobs as an RNase in RNA silencing pathway and death-jobs as a DNase in PCD pathway. Specifically, we will test the hypothesis that cleavage of the human Dicer by pro-apoptotic caspase-3 produces a stable C-terminal fragment of Dicer (Dcr-C) that functions as a DNase (DNA nickase) in the PCD pathway in human cells. We will also test the hypothesis that the conversion of Dicer to Dcr-C and its DNA nickase activity are crucial for anti-viral immunity. Our exciting preliminary studies showed that Dcr-C exhibits DNA nickase activity in test tube and in cells, supporting our hypotheses. If successful, the proposed studies employing biochemical and cellular approaches will form foundations for future animal studies of Dicer RNase-to-DNase conversion in PCD and anti-viral immunity in mammals. Understanding the physiological roles and molecular mechanisms of Dcr- C DNA nickase will reveal new regulatory mechanisms in the PCD pathway and anti-viral system including a novel link between RNA silencing and PCD via Dicer, both of which are crucial for anti-viral defense.