Insights into metalloregulation by M-box riboswitch RNAs via structural analysis of manganese-bound complexes

The M-box riboswitch couples intracellular magnesium levels to expression of bacterial metal transport genes. Structural analyses on other riboswitch RNA classes, which typically respond to a small organic metabolite, have revealed that ligand recognition occurs through a combination of base-stacking, electrostatic, and hydrogen-bonding interactions. In contrast, the M-box RNA triggers a change in gene expression upon association with an undefined population of metals, rather than responding to only a single ligand. Prior biophysical experimentation suggested that divalent ions associate with the M-box RNA to promote a compacted tertiary conformation, resulting in sequestration of a short sequence tract otherwise required for downstream gene expression. Electrostatic shielding from loosely associated metals is undoubtedly an important influence during this metal-mediated compaction pathway. However, it is also likely that a subset of divalent ions specifically occupies cation binding sites and promotes proper positioning of functional groups for tertiary structure stabilization. To better elucidate the role of these metal binding sites, we resolved a manganese-chelated M-box RNA complex to 1.86 Å by X-ray crystallography. These data support the presence of at least eight well-ordered cation binding pockets, including several sites that had been predicted by biochemical studies but were not observed in prior structural analysis. Overall, these data support the presence of three metal-binding cores within the M-box RNA that facilitate a network of long-range interactions within the metal-bound, compacted conformation.     

Rise of the RNA Machines: Exploring the Structure of Long Non-Coding RNAs

Novel, profound and unexpected roles of long non-coding RNAs (lncRNAs) are emerging in critical aspects of gene regulation. Thousands of lncRNAs have been recently discovered in a wide range of mammalian systems, related to development, epigenetics, cancer, brain function and hereditary disease. The structural biology of these lncRNAs presents a brave new RNA world, which may contain a diverse zoo of new architectures and mechanisms. While structural studies of lncRNAs are in their infancy, we describe existing structural data for lncRNAs, as well as crystallographic studies of other RNA machines and their implications for lncRNAs. We also discuss the importance of dynamics in RNA machine mechanism. Determining commonalities between lncRNA systems will help elucidate the evolution and mechanistic role of lncRNAs in disease, creating a structural framework necessary to pursue lncRNA-based therapeutics.     

Multi-domain Packing in the Aminoacylatable 3′ End of a Plant Viral RNA

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure (TLS) in its 3′ untranslated region (3′ UTR).  This highly structured element induces valylation of the viral RNA by host cell enzymes and is important for virus proliferation. Directly upstream of the TYMV TLS is an upstream pseudoknot domain (UPD) that has been considered to be structurally distinct from the TLS.  However, using a combination of functional, biochemical, and biophysical assays, we show that the entire 3′ UTR of the viral genome is a single structured element in the absence of cellular protein.  This packing architecture stabilizes the RNA structure and creates a better substrate for aminoacylation, and thus the UPD and TLS are functionally and structurally coupled.  It has been proposed that the TYMV TLS acts as a molecular switch between translation and replication. Our results suggest that this putative switch could be based on structural changes within the global architecture of the UTR induced by interactions with the ribosome. The TYMV TLS·UPD might demonstrate how RNA structural plasticity can play a role in regulation of biological processes.     

Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes

Bark beetles oxidize the defensive monoterpenes of their host trees both to detoxify them and convert them into components of their pheromone system. This oxidation is catalyzed by cytochrome P450 enzymes and occurs in different tissues of the insect, including the gut (i.e., the site where the beetle's pheromones are produced and accumulated) and the antennae (i.e., the olfactory organs used for perception of airborne defensive monoterpenes as well as other host-associated compounds and pheromones). We identified ten new CYP genes in the pine beetle Dendroctonus rhizophagus in either antennae or gut tissue after stimulation with the vapors of major host monoterpenes α-pinene, β-pinene and 3-carene. Five genes belong to the CYP4 family, four to the CYP6 family and one to the CYP9 family. Differential expression of almost all of the CYP genes was observed between sexes, and within these significant differences among time, stimuli, anatomical region, and their interactions were found upon exposure to host monoterpenes. Increased expression of cytochrome P450 genes suggests that they play a role in the detoxification of monoterpenes released by this insect's host trees.