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Background: Adenoviruses use the short non-coding transcript VA RNA I to inhibit host antiviral kinase PKR. Results: VA RNA I contains a pH-and Mg 2ϩ -sensitive tertiary structure that, unexpectedly, is not required for PKR inhibition. Conclusion: Structural requirements for an RNA inhibitor of PKR are simpler than appreciated previously. Significance: These findings explain how non-coding RNAs of varied sequence and structure can efficiently inhibit PKR.

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.

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.

Chemical and enzymatic RNA structure probing methods are important tools for examining RNA secondary and tertiary structures and their interactions with proteins, small molecules, and ions. The recently developed "Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension" (SHAPE) approach has proven especially useful for uncovering details of secondary structures, complex tertiary interactions, and RNA dynamics. Analysis of short RNAs using SHAPE or other probing methods that require reverse transcription to detect RNA modifications presents a technical hurdle in that intense bands corresponding to abortive transcription during primer extension and the full-length RT product may obscure information corresponding to the 3' and 5' ends of the molecule, respectively. This chapter describes the design and use of an RNA "structure cassette" that addresses these issues. First, we describe methods by which any RNA of interest may be cloned into a new plasmid preloaded with sequences that encode a structure cassette surrounding the short internal target RNA. Next, we outline key considerations and analyses of the RNAs produced that should be performed prior to SHAPE or other structure probing experiments.