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. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus.

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. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity.

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. In this chapter, we detail this method using the titration of an adenine-binding RNA with 2,6-diaminopurine (DAP) as a practical example. Our insights should be generally applicable to observing the interactions of a broad range of molecules with structured RNAs.

Computational and analytical framework for small RNA profiling by high-throughput sequencing.

The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.

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. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173-AGO1 complex possesses unique functionality that many other miRNA-AGO1 complexes lack.

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. Analysis of the temperature-dependent reactivity of the RNA in the presence and absence of hypoxanthine reveals that a limited set of nucleotides within the binding pocket change their conformation in response to ligand binding. Our data demonstrate that a distal loop-loop interaction serves to restrict the conformational freedom of a significant portion of the three-way junction, thereby promoting ligand binding under physiological conditions.

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.

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. Notably, this is the first structure of a complete riboswitch containing all sequences associated with both the ligand binding aptamer domain and the regulatory expression platform. Chemical probing of this RNA in the absence and presence of ligand shows how the structure changes in response to S-adenosylmethionine to sequester the ribosomal binding site and affect translational gene regulation.

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. To understand the contribution of conserved nucleotides in both the three-way junction and the loop-loop interaction of this RNA, we performed a detailed mutagenic survey of these elements in the context of an adenine-responsive variant of the xpt-pbuX guanine riboswitch from Bacillus subtilis. The varying ability of these mutants to bind ligand as measured by isothermal titration calorimetry uncovered the conserved nucleotides whose identity is required for purine binding. Crystallographic analysis of the bound form of five mutants and chemical probing of their free state demonstrate that the identity of several universally conserved nucleotides is not essential for formation of the RNA-ligand complex but rather for maintaining a binding-competent form of the free RNA. These data show that conservation patterns in riboswitches arise from a combination of formation of the ligand-bound complex, promoting an open form of the free RNA, and participating in the secondary structural switch with the expression platform.

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. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.

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. Here we report the crystal structure at 1.95 A resolution of the purine-binding domain of the guanine riboswitch from the xpt-pbuX operon of B. subtilis bound to hypoxanthine, a prevalent metabolite in the bacterial purine salvage pathway. This structure reveals a complex RNA fold involving several phylogenetically conserved nucleotides that create a binding pocket that almost completely envelops the ligand. Hypoxanthine functions to stabilize this structure and to promote the formation of a downstream transcriptional terminator element, thereby providing a mechanism for directly repressing gene expression in response to an increase in intracellular concentrations of metabolite.