STRUCTURAL AND BIOCHEMICAL CHARACTERIZATIONS OF THE INNATE IMMUNE SENSOR/EFFECTOR RNASE L

Abstract

One of the defensive mechanisms activated by type I interferons in human tissues involves synthesis of 2', 5'-linked oligoadenylates (2-5As) by the oligoadenylate synthetase (OAS) family members and cleavage of intracellular RNA by the 2-5A-dependent kinase-like endoribonuclease, RNase L. Despite its important roles that extend from innate immunity, the detailed mechanism of activation and cleavage-site selection of RNase L remained poorly known. Here, I have combined X-ray crystallography and biochemical studies to characterize RNase L in solution, elucidate its three-dimensional architecture in the presence of both 2-5A and RNA substrate, and explain its behavior upon activation.

Our kinetic measurements, cross-linking assays, and size-exclusion chromatography analyses all converge to suggest that human RNase L forms high-order self-assemblies when reaching its fully active state, similar to the mode of activation of the phylogenetically related transmembrane kinase/RNase Ire1. The structural analysis of the N-terminal ankyrin-repeat (ANK) domain of RNase L in the presence and absence of 2-5A reveals that two molecules of 2-5A simultaneously tether two ANK domains to form a head-to-tail dimeric complex. Contrary to previous beliefs, this process does not involve any structural changes in the ANK domain.

To better understand domain interactions of RNase L, we have further obtained two crystal structures of nearly full-length human RNase L in complexes with synthetic or natural 2-5A, adenine nucleotides, and, for the first time, a fragment of RNA. Overall, RNase L forms a crossed homodimer by exchanging ANK domains, and positions two RNase domains for recognition and cleavage of RNA. Combined with corroborating biochemical studies, the partial RNA observed in the active site allowed us to unveil the UN^N (^ denotes the cleavage site) rule that RNase L follows to select cleavage sites: one protomer of RNase L recognizes the identity nucleotide uridine, whereas the other protomer cleaves RNA between the two nucleotides immediately following the uridine. This mechanism readily explains the requirement of 2-5A-induced dimerization of RNase L for its activation. Furthermore, the UN^N rule is consistent with the UG^C cleavage pattern of Ire1, highlighting similarities between substrate selection and cleavage mechanisms employed by RNase L and Ire1.