Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis.

RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1.

Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs.

Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand.

Monitoring RNA-ligand interactions using isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is a biophysical technique that measures the heat evolved or absorbed during a reaction to report the enthalpy, entropy, stoichiometry of binding, and equilibrium association constant. A significant advantage of ITC over other methods is that it can be readily applied to almost any RNA-ligand complex without having to label either molecule and can be performed under a broad range of pH, temperature, and ionic concentrations. During our application of ITC to investigate the thermodynamic details of the interaction of a variety of compounds with the purine riboswitch, we have explored and optimized experimental parameters that yield the most useful and reproducible results for RNAs.

AGO1-miR173 complex initiates phased siRNA formation in plants.

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation-cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway.

Ligand-dependent folding of the three-way junction in the purine riboswitch.

Riboswitches are highly structured cis-acting elements located in the 5'-untranslated region of messenger RNAs that directly bind small molecule metabolites to regulate gene expression. Structural and biochemical studies have revealed riboswitches experience significant ligand-dependent conformational changes that are coupled to regulation. To monitor the coupling of ligand binding and RNA folding within the aptamer domain of the purine riboswitch, we have chemically probed the RNA with N-methylisatoic anhydride (NMIA) over a broad temperature range.

Structure of the SAM-II riboswitch bound to S-adenosylmethionine.

In bacteria, numerous genes harbor regulatory elements in the 5' untranslated regions of their mRNA, termed riboswitches, which control gene expression by binding small-molecule metabolites. These sequences influence the secondary and tertiary structure of the RNA in a ligand-dependent manner, thereby directing its transcription or translation. The crystal structure of an S-adenosylmethionine-responsive riboswitch found predominantly in proteobacteria, SAM-II, has been solved to reveal a second means by which RNA interacts with this important cellular metabolite.

The Cech Symposium: a celebration of 25 years of ribozymes, 10 years of TERT, and 60 years of Tom.

The Cech Symposium was held in Boulder, Colorado, on July 12-13, 2007, to celebrate a triple anniversary: 25 years since the first publication reporting RNA self-splicing, 10 years since the identification of reverse transcriptase motifs in the catalytic subunit of telomerase, and 60 years since the birth of Thomas R. Cech. Past and present members of the Cech laboratory presented on their current research, which branched into many categories of study including RNA-mediated catalysis, telomerase and telomeres, new frontiers in nucleic acids, alternative splicing, as well as scientific research with direct medical applications.

Mutational analysis of the purine riboswitch aptamer domain.

The purine riboswitch is one of a number of mRNA elements commonly found in the 5'-untranslated region capable of controlling expression in a cis-fashion via its ability to directly bind small-molecule metabolites. Extensive biochemical and structural analysis of the nucleobase-binding domain of the riboswitch, referred to as the aptamer domain, has revealed that the mRNA recognizes its cognate ligand using an intricately folded three-way junction motif that completely encapsulates the ligand. High-affinity binding of the purine nucleobase is facilitated by a distal loop-loop interaction that is conserved between both the adenine and guanine riboswitches.

Modified pyrimidines specifically bind the purine riboswitch.

The purine riboswitch is a genetic regulatory element found in the 5'-untranslated regions of Gram-positive bacteria that regulates expression of the mRNA specifically in response to either guanine or adenine. We report that the adenine-responsive RNA element is also capable of specifically recognizing pyrimidine compounds bearing modifications at the 6- or 5,6-positions in a fashion similar to that of purine compounds. Using isothermal titration calorimetry and X-ray crystallography, the binding of these compounds is characterized.

Riboswitches: fold and function.

Riboswitches in the 5'-untranslated regions of mRNAs cotranscriptionally couple ligand binding and gene regulation. In this issue of Chemistry & Biology, Lemay et al. describe folding of a key tertiary interaction in the adenine riboswitch and its mechanistic consequences.

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain.

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine.

Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine.

Riboswitches are genetic regulatory elements found in the 5' untranslated region of messenger RNA that act in the absence of protein cofactors. They are broadly distributed across bacteria and account for the regulation of more than 2% of all genes in Bacillus subtilis, underscoring their importance in the control of cellular metabolism. The 5' untranslated region of many mRNAs of genes involved in purine metabolism and transport contain a guanine-responsive riboswitch that directly binds guanine, hypoxanthine or xanthine to terminate transcription.