A novel RNA molecular signature for activation of 2'-5' oligoadenylate synthetase-1.

Human 2'-5' oligoadenylate synthetase-1 (OAS1) is central in innate immune system detection of cytoplasmic double-stranded RNA (dsRNA) and promotion of host antiviral responses. However, the molecular signatures that promote OAS1 activation are currently poorly defined. We show that the 3'-end polyuridine sequence of viral and cellular RNA polymerase III non-coding transcripts is critical for their optimal activation of OAS1. Potentiation of OAS1 activity was also observed with a model dsRNA duplex containing an OAS1 activation consensus sequence. We determined that the effect is attributable to a single appended 3'-end residue, is dependent upon its single-stranded nature with strong preference for pyrimidine residues and is mediated by a highly conserved OAS1 residue adjacent to the dsRNA binding surface. These findings represent discovery of a novel signature for OAS1 activation, the 3'-single-stranded pyrimidine (3'-ssPy) motif, with potential functional implications for OAS1 activity in its antiviral and other anti-proliferative roles.

Vachon VK ，Calderon BM ，Conn GL 《-》

Human OAS1 activation is highly dependent on both RNA sequence and context of activating RNA motifs.

2'-5'-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2'-5'-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3'-end single-stranded pyrimidine (3'-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.

Schwartz SL ，Park EN ，Vachon VK ，Danzy S ，Lowen AC ，Conn GL ... -  《-》

A human cellular noncoding RNA activates the antiviral protein 2'-5'-oligoadenylate synthetase 1.

The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes sense cytosolic dsRNA, a potent signal of viral infection. In response to dsRNA binding, OAS proteins synthesize the second messenger 2'-5'-linked oligoadenylate that activates the latent ribonuclease L (RNase L). RNase L-mediated degradation of viral and cellular RNAs effectively halts viral replication and further stimulates innate immune responses by inducing type I interferon. The OAS/RNase L pathway is therefore central in innate immune recognition and promotion of antiviral host responses. However, the potential for specific RNA sequences or structures to drive OAS1 activation and the molecular mechanisms by which they act are not currently fully understood. Moreover, the cellular regulators of OAS activity are not well defined. Here, we demonstrate that the human cellular noncoding RNA 886 (nc886) activates OAS1 both in vitro and in human A549 cells. We show that a unique structure present only in one of the two structural conformers adopted by nc886 drives potent OAS1 activation. In contrast, the conformer lacking this unique structure activated OAS1 only very weakly. We also found that formation of this OAS1-activating structural motif depends on the nucleotides in the apical-most loop of nc886 and the adjacent helix. These findings identify a cellular RNA capable of activating the OAS/RNase L pathway in human cells and illustrate the importance of structural elements, and their context, in potentiating OAS1 activity.

Calderon BM ，Conn GL 《-》

Role of helical structure and dynamics in oligoadenylate synthetase 1 (OAS1) mismatch tolerance and activation by short dsRNAs.

Schwartz SL ，Dey D ，Tanquary J ，Bair CR ，Lowen AC ，Conn GL ... -  《-》

The Activation Mechanism of 2'-5'-Oligoadenylate Synthetase Gives New Insights Into OAS/cGAS Triggers of Innate Immunity.

2'-5'-Oligoadenylate synthetases (OASs) produce the second messenger 2'-5'-oligoadenylate, which activates RNase L to induce an intrinsic antiviral state. We report on the crystal structures of catalytic intermediates of OAS1 including the OAS1·dsRNA complex without substrates, with a donor substrate, and with both donor and acceptor substrates. Combined with kinetic studies of point mutants and the previously published structure of the apo form of OAS1, the new data suggest a sequential mechanism of OAS activation and show the individual roles of each component. They reveal a dsRNA-mediated push-pull effect responsible for large conformational changes in OAS1, the catalytic role of the active site Mg(2+), and the structural basis for the 2'-specificity of product formation. Our data reveal similarities and differences in the activation mechanisms of members of the OAS/cyclic GMP-AMP synthase family of innate immune sensors. In particular, they show how helix 3103-α5 blocks the synthesis of cyclic dinucleotides by OAS1.

Lohöfener J ，Steinke N ，Kay-Fedorov P ，Baruch P ，Nikulin A ，Tishchenko S ，Manstein DJ ，Fedorov R ... -  《-》