SUMMARY
The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic-GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here we identify natural variants of human STING that are poorly responsive to cGAMP, yet unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a non-canonical cyclic-di-nucleotide, cyclic[G(2′ -5′)pA(3′ -5′)p], which contains a single 2′ -5′ phosphodiester bond. Cyclic[G(2′ -5′)pA(3′ -5′)p] potently activates diverse human STING receptors and may therefore be a useful adjuvant or immunotherapeutic. Our results indicate that human STING variants have evolved that can distinguish conventional (3′ -5′) cyclic-di-nucleotides, known only to be produced by bacteria, from the non-canonical cyclic-di-nucleotide produced by mammalian cGAS.
Tumor necrosis factor-α (TNF-α) is the most potent proinflammatory cytokine in mammals. The degradation of TNF-α mRNA is critical for restricting TNF-α synthesis and involves a constitutive decay element (CDE) in the 3' UTR of the mRNA. Here, we demonstrate that the CDE folds into an RNA stem-loop motif that is specifically recognized by Roquin and Roquin2. Binding of Roquin initiates degradation of TNF-α mRNA and limits TNF-α production in macrophages. Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE sequences are highly conserved and are found in more than 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, CDE-containing mRNAs were identified as the primary targets of Roquin on a transcriptome-wide scale. Thus, Roquin proteins act broadly as mediators of mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs.
Cyclic di-nucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed fluorescent RNA-based biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic di-nucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.
Riboswitches are structured RNAs typically located in the 5' untranslated regions of bacterial mRNAs that bind metabolites and control gene expression. Most riboswitches sense one metabolite and function as simple genetic switches. However, we found that the 5' region of the Bacillus clausii metE messenger RNA includes two riboswitches that respond to S-adenosylmethionine and coenzyme B12. This tandem arrangement yields a composite gene control system that functions as a two-input Boolean NOR logic gate. These findings and the discovery of additional tandem riboswitch architectures reveal how simple RNA elements can be assembled to make sophisticated genetic decisions without involving protein factors.
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