A scientific journey through the 2-5A/RNase L system

Abstract

The antiviral and antitumor actions of interferons are caused, in part, by a remarkable regulated RNA cleavage pathway known as the 2-5A/RNase L system. 2'-5' linked oligoadenylates (2-5A) are produced from ATP by interferon-inducible synthetases. 2-5A activates pre-existing RNase L, resulting in the cleavage of RNAs within single-stranded regions. Activation of RNase L by 2-5A leads to an antiviral response, although precisely how this happens is a subject of ongoing investigations. Recently, RNase L was identified as the hereditary prostate cancer 1 gene. That finding has led to the discovery of a novel human retrovirus, XMRV. My scientific journey through the 2-5A system recounts some of the highlights of these efforts. Knowledge gained from studies on the 2-5A system could have an impact on development of therapies for important viral pathogens and cancer.

Figure 1. The 2-5A/RNase L pathway is a classic antiviral innate immune pathway

The viral pathogen associated molecular pattern, dsRNA, activates IFN induced 2-5A synthetase (also known as oligoadenylate synthetase or OAS). This results in the synthesis of 2-5A from ATP. The 2-5A bindings to inactive monomeric RNase L, forcing the formation of activated dimers of RNase L. The resulting degradation of single stranded loop regions in RNA, including rRNA in intact ribosomes, produces a potent antiviral response in the IFN treated and virus infected cell.

Figure 2. Cloning of RNase L with radiolabeled 2-5A as probe (as described in ref. [26])

(a-c) Post-mitochondrial supernatant fraction of mouse L cells was used to write the word “2-5A” on nitrocellulose filters. Subsequently, the filters were incubated in a solution of the 2-5A probe (a) without additions, (b) with unlabeled 2-5A as competitor, and (c) with unlabeled ATP. The filters were washed, dried and used to expose x-ray film. The results on autoradiography show that binding was prevented with unlabeled 2-5A but not with ATP, thus demonstrating the feasibility of the approach. The first clone for a partial murine RNase L was obtained by screening a cDNA expression library of IFN and cycloheximide treated mouse L cells. The third round screening was done (d) without competitor, (e) with unlabeled 2-5A as competitor, or (f) with unlabeled ATP.

Figure 3. The domain structure of RNase L

Ank, ankyrin repeat; Ire1homology, nuclease domain similar to Ire1 kinase/endoribonucleases.