G-quadruplexes (G4s) are four-stranded nucleic acid structures that arise from the stacking of Gquartets, cyclic arrangements of four guanines engaged in Hoogsteen base pairing. Until recently, most RNA G4 structures were thought to conform to a sequence pattern in which guanines stacking within the G4 would also be contiguous in sequence (e.g., four successive guanine trinucleotide tracts separated by loop nucleotides). Such a sequence restriction, and the stereochemical constraints inherent to RNA (arising, in particular, from presence of the 2'-OH), dictate relatively simple RNA G4 structures. Recent crystallographic and solution NMR structure determinations of a number of in vitro selected RNA aptamers has revealed RNA G4 structures of unprecedented complexity. Structures of the Sc1 aptamer that binds an RGG peptide from the Fragile-X Mental Retardation protein, various fluorescence turn-on aptamers (Corn, Mango, and Spinach), and the spiegelmer that binds the complement protein C5a, in particular, reveal complexity hitherto unsuspected in RNA G4s, including nucleotides in syn conformation, locally inverted strand polarity, and nucleotide quartets that are not all-G. Common to these new structures, the sequences folding into G4s do not conform to the requirement that guanine stacks arise from consecutive (contiguous in sequence) nucleotides. This review highlights how emancipation from this constraint drastically expands the structural possibilities of RNA G-quadruplexes.
The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of C9orf72 RAN translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially binds to the repeat RNA’s G-quadruplex conformation, and efficiently unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with the G4C2 repeat RNA and is essential for effective RAN translation in the cell. In induced pluripotent stem cells and differentiated motor neurons derived from C9orf72-linked ALS patients, reducing DHX36 significantly decreased the levels of endogenous DPR proteins. DHX36 is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex structures and may be a potential target for therapeutic intervention.
MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple protontransfer events. Of particular interest is the protonation state of residue D198, which possesses a pK a above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wildtype HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.neutron diffraction | enzyme mechanism | proton transfer | nucleosidase | Helicobacter
S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) are an essential superfamily of enzymes that catalyze the transfer of a methyl group to several biomolecules. Alterations in the methylation of cellular components crucially impact vital biological processes, making MTases attractive drug targets for treating infectious diseases and diseases caused by overactive human-encoded MTases. Several methods have been developed for monitoring the activity of MTases, but most MTase assays have inherent limitations or are not amenable for high-throughput screening. We describe a universal, competitive fluorescence polarization (FP) assay that directly measures the production of S-adenosylhomocysteine (AdoHcy) from MTases. Our developed assay monitors the generation of AdoHcy by displacing a fluorescently labeled AdoHcy molecule complexed to a catalytically inert 5′-methylthioadenosine nucleosidase (MTAN-D198N) variant performed in a mix-and-read format. Producing the fluorescently labeled molecule involves a one-pot synthesis by combining AdoHcy with an amine-reactive rhodamine derivative, which possesses a Kd value of 11.3 ± 0.7 nM to MTAN-D198N. The developed competitive FP assay expresses a limit of detection for AdoHcy of 6 nM and exhibits a 34-fold preference to AdoHcy in comparison to AdoMet. We demonstrate the utility of the developed assay by performing a pilot screen with the NIH Clinical Collection as well as determining the kinetic parameters of l-histidine methylation for EgtD from Mycobacterium tuberculosis. Additionally, the developed assay is applicable to other AdoMet-dependent and ATP-dependent enzymes by detecting various adenosine-containing molecules including 5′-methylthioadenosine, AMP, and ADP.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.