SUMMARY The ZTP riboswitch is a widespread family of regulatory RNAs that upregulate de novo purine synthesis in response to increased intracellular levels of ZTP or ZMP (AICAR). As an important intermediate in purine biosynthesis, ZMP also serves as a proxy for the concentration of 10-formyltetrahydrofolate, a key component of one carbon metabolism. Here we report the structure of the ZTP riboswitch bound to ZMP at a resolution of 1.80 Å. The RNA contains two subdomains brought together through a long-range pseudoknot further stabilized through helix-helix packing. ZMP is bound at the subdomain interface of the RNA through a set of interactions with the ligand's base, ribose sugar and phosphate moieties. Unique to nucleobase recognition by RNAs, the Z base is inner sphere coordinated to a magnesium cation bound by two backbone phosphates. This interaction, along with steric hindrance by the backbone, imparts specificity over related analogs such as ATP/AMP.

ObjectiveTemporal lobe epilepsy (TLE) patients exhibit signs of memory impairments even when seizures are pharmacologically controlled. Surprisingly, the underlying molecular mechanisms involved in TLE-associated memory impairments remain elusive. Memory consolidation requires epigenetic transcriptional regulation of genes in the hippocampus; therefore, we aimed to determine how epigenetic DNA methylation mechanisms affect learning-induced transcription of memory-permissive genes in the epileptic hippocampus.MethodsUsing the kainate rodent model of TLE and focusing on the brain-derived neurotrophic factor (Bdnf) gene as a candidate of DNA methylation-mediated transcription, we analyzed DNA methylation levels in epileptic rats following learning. After detection of aberrant DNA methylation at the Bdnf gene, we investigated functional effects of altered DNA methylation on hippocampus-dependent memory formation in our TLE rodent model.ResultsWe found that behaviorally driven BdnfDNA methylation was associated with hippocampus-dependent memory deficits. Bisulfite sequencing revealed that decreased BdnfDNA methylation levels strongly correlated with abnormally high levels of BdnfmRNA in the epileptic hippocampus during memory consolidation. Methyl supplementation via methionine (Met) increased BdnfDNA methylation and reduced BdnfmRNA levels in the epileptic hippocampus during memory consolidation. Met administration reduced interictal spike activity, increased theta rhythm power, and reversed memory deficits in epileptic animals. The rescue effect of Met treatment on learning-induced BdnfDNA methylation, Bdnf gene expression, and hippocampus-dependent memory, were attenuated by DNA methyltransferase blockade.InterpretationOur findings suggest that manipulation of DNA methylation in the epileptic hippocampus should be considered as a viable treatment option to ameliorate memory impairments associated with TLE.

A recent bioinformatic analysis of well-characterized classes of riboswitches uncovered subgroups unable to bind to the regulatory molecule of the parental class. Within the guanine/adenine class, seven groups of RNAs were identified that deviate from the consensus sequence at one or more of three positions directly involved purine nucleobase recognition, one of which was validated as a second class of 2′-deoxyguanosine riboswitch (called 2′-dG-II). To understand how 2′-dG-II riboswitches recognize their cognate ligand and how they differ from a previously identified class of 2′-deoxyguanosine binding riboswitches, we have solved the crystal structure of a 2′-dG-II aptamer domain bound to 2′-deoxyguanosine. This structure reveals a global architecture similar to other members of the purine riboswitch family, but contains key differences within the ligand binding core. Defining the 2′-dG-II riboswitches is a two-nucleotide insertion in the three-way junction that promotes novel base-base interactions. Unlike 2′-dG-I riboswitches, the 2′-dG-II class only requires local changes to the ligand binding pocket of the guanine/adenine class to achieve a change in ligand preference. Notably, members of the 2′-dG-II family have variable ability to discriminate between 2′-deoxyguanosine and riboguanosine, suggesting that a subset of 2′-dG-II riboswitches may bind either molecule to regulate gene expression.

Riboswitches are important model systems for the development of approaches to search for RNA-targeting therapeutics. A principal challenge in finding compounds that target riboswitches is that the effector ligand is typically almost completely encapsulated by the RNA, which severely limits the chemical space that can be explored. Efforts to find compounds that bind the guanine/adenine class of riboswitches with a high affinity have in part focused on purines modified at the C6 and C2 positions. These studies have revealed compounds that have low to sub-micromolar affinity and, in a few cases, have antimicrobial activity. To further understand how these compounds interact with the guanine riboswitch, we have performed an integrated structural and functional analysis of representative guanine derivatives with modifications at the C8, C6 and C2 positions. Our data indicate that while modifications of guanine at the C6 position are generally unfavorable, modifications at the C8 and C2 positions yield compounds that rival guanine with respect to binding affinity. Surprisingly, C2-modified guanines such as N2-acetylguanine completely disrupt a key Watson–Crick pairing interaction between the ligand and RNA. These compounds, which also modulate transcriptional termination as efficiently as guanine, open up a significant new chemical space of guanine modifications in the search for antimicrobial agents that target purine riboswitches.

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