Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5′end. The exact structure of RNA activating RIG-I remains controversial. Here we established a chemical approach for 5′triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5′triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double strand conformation with base pairing of the nucleoside carrying the 5′triphosphate was required. RIG-I activation was impaired by a 3′overhang at the 5′triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative strand RNA viruses which lack long double-stranded RNA but do contain panhandle blunt short double-stranded 5′triphosphate RNA in their single-stranded genome.

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, whose PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. We report on structures of ternary complexes of T. thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nt guide DNA and complementary target RNAs of length 12-, 15- and 19-nt, which define the molecular basis for Mg2+-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of helix requiring release of the 3′-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate backbone modified target strands revealed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

Here we report on a 3.0 Å crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ-and PIWI-containing lobes of argonaute widens on formation of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2′-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.RNA-induced silencing complex (RISC)-associated argonaute (Ago) proteins composed of PAZ-and PIWI-containing modules have a central role in mediating distinct assembly and cleavage steps of the RNA interference (RNAi) catalytic cycle [1][2][3][4] . The Ago protein, as the sole component of RISC exhibiting RNA 'slicer' activity [5][6][7] , is a critical player in the RNAi pathway 8 , effecting transcriptional and post-transcriptional gene regulation in plants and animals [1][2][3][4] . In this capacity, Agos have essential roles ranging from maintaining genomic integrity to heterochromatin formation. Some Ago proteins with active endonuclease domains contribute to the maturation of bound short interfering RNAs (siRNAs) by degradative cleavage of the passenger strand and subsequent guide-strand-mediated sequence-specific ©2008 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to D.J.P. (pateld@mskcc.org) or T.T. (ttuschl@mail.rockefeller.edu). Author Contributions Y.W. and G.S. expressed and purified T. thermophilus Ago, and grew crystals of the ternary complex. H.L. and Y.W. collected X-ray diffraction data on the micro-focus beam line, and Y.W. solved the structure of the ternary complex. The structural studies were undertaken with the supervision of D.J.P. S.J. was responsible for the cleavage assays on Ago with modified guide strands under the supervision of T.T. D.J.P. and T.T. were primarily responsible for writing the paper and all authors read and approved the submitted manuscript. Author InformationThe structural coordinates of the ternary complex of T. thermophilus Ago bound to 5′-phosphorylated 21-nucleotide guide DNA and 20-nucleotide target RNA have been submitted to the Protein Data Bank under accession number 3F73. Reprints and permissions information is available at www.nature.com/reprints.Supplementary Information is linked to the online version of...

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5′-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson–Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5′-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3′-end-binding pocket made up of the PAZ domain show little effect.

Several distinct classes of small RNAs, some newly identified, have been discovered to play important regulatory roles in diverse cellular processes. These classes include siRNAs, miRNAs, rasiRNAs and piRNAs. Each class binds to distinct members of the Argonaute/Piwi protein family to form ribonucleoprotein complexes that recognize partially, or nearly perfect,complementary nucleic acid targets, and that mediate a variety of regulatory processes, including transcriptional and post-transcriptional gene silencing. Based on the known relationship of Argonaute/Piwi proteins with distinct classes of small RNAs, we can now predict how many new classes of small RNAs or silencing processes remain to be discovered.

RIG-I is a cytosolic helicase that senses 5’-ppp-RNA contained in negative strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies establish that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5’-ppp-RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD domain bound to both blunt-ends of a self-complementary 5’-ppp-dsRNA 12-mer, with interactions involving 5’-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters the observable 5’-pp of the bound RNA, with a stacked Phe capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5’-ppp-dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2’-OCH3 RNA modifications on the interferon response.

DNA and RNA can fold into a variety of alternative conformations. In recent years, a particular nucleic acid structure was discussed to play a role in malignant transformation and cancer development. This structure is called a G-quadruplex (G4). G4 structure formation can drive genome instability by creating mutations, deletions and stimulating recombination events. The importance of G4 structures in the characterization of malignant cells was currently demonstrated in breast cancer samples. In this analysis a correlation between G4 structure formation and an increased intratumor heterogeneity was identified. This suggests that G4 structures might allow breast cancer stratification and supports the identification of new personalized treatment options. Because of the stability of G4 structures and their presence within most human oncogenic promoters and at telomeres, G4 structures are currently tested as a therapeutic target to downregulate transcription or to block telomere elongation in cancer cells. To date, different chemical molecules (G4 ligands) have been developed that aim to target G4 structures. In this review we discuss and compare G4 function and relevance for therapeutic approaches and their impact on cancer development for three cancer entities, which differ significantly in their amount and type of mutations: pancreatic cancer, leukemia and malignant melanoma. G4 structures might present a promising new strategy to individually target tumor cells and could support personalized treatment approaches in the future.

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress.