ADARs (adenosine deaminases acting on RNA) are editing enzymes that convert adenosine (A) to inosine (I) in duplex RNA, a modification reaction with wide-ranging consequences on RNA function. Our understanding of the ADAR reaction mechanism, origin of editing site selectivity and effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the deaminase domain of human ADAR2 bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis for ADAR deaminase domain’s dsRNA specificity, its base-flipping mechanism, and nearest neighbor preferences. In addition, an ADAR2-specific RNA-binding loop was identified near the enzyme active site rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

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It is widely thought that Alzheimer's disease (AD) begins as a malfunction of synapses, eventually leading to cognitive impairment and dementia. Homeostatic synaptic scaling is a mechanism that could be crucial at the onset of AD but has not been examined experimentally. In this process, the synaptic strength of a neuron is modified so that the overall excitability of the cell is maintained. Here, we investigate whether synaptic scaling mediated by L-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) contributes to pathology in double knockin (2؋KI) mice carrying human mutations in the genes for amyloid precursor protein and presenilin-1. By using whole-cell recordings, we show that 2؋KI mice exhibit age-related downscaling of AMPARmediated evoked currents and spontaneous, miniature currents. Electron microscopic analysis further corroborates the synaptic AMPAR decrease. Additionally, 2؋KI mice show age-related deficits in bidirectional plasticity (long-term potentiation and longterm depression) and memory flexibility. These results suggest that AMPARs are important synaptic targets for AD and provide evidence that cognitive impairment may involve downscaling of postsynaptic AMPAR function.amyloid precursor protein ͉ glutamate ͉ presenilin E xtensive work on Alzheimer's disease (AD) has led to the hypothesis that the memory failure exhibited by patients in the early stages of AD results from synaptic disruption (1-3), without frank neuronal loss, which is caused by toxicity of the 42-aa variant of the amyloid ␤ protein (A␤ 42 ). Work in AD models (4) shows that A␤ 42 impairs synaptic plasticity in brain regions, such as the hippocampus, that are recognized early targets for AD. Transgenic (Tg) mice with familial AD mutations display disruptions of long-term potentiation (LTP), an electrophysiological correlate of memory encoding (5). The LTP impairment occurs before deposition of A␤ plaques (4, 6), making it a sensitive marker for early AD dysfunction. Notably, the late phase of LTP (called expression) is highly susceptible to disruption (6-9). LTP expression relies on alterations of L-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors (AMPARs), including phosphorylation by kinases and recruitment of AMPARs to the synaptic membrane (10). Conversely, AMPAR removal is thought to mediate the activitydependent decrease of excitatory transmission (11), which is elicited by the paradigm of long-term depression (LTD) (12). Importantly, LTD has been scarcely studied in AD models (4, 13), particularly Tg-AD mice.Recently, AMPARs have been implicated in a slower form of synaptic plasticity, termed homeostatic synaptic scaling, in which the total synaptic strength of a neuron is modified to regulate its excitability (14). Synaptic scaling involves, among other factors, the postsynaptic insertion and removal of AMPARs and changes in the turnover rate of functional receptors (14-16). Adjustments by synaptic scaling operate in vivo (17) and seem critical for regulating synaptic strength during lear...

SynopsisThe base-stacking patterns in over 70 published crystal structures of nucleic acid constituents and polynucleotides were examined. Several recurring stacking patterns were found. Base stacking in the solid state apparently is very specific, with particular modes of interaction persisting in various crystalline environments. The vertical stacking of purines and pyrimidines in polynucleotides is similar to that observed in crystals of tiucleic acid constituents. Only partial base overlap was found in the majority of the structures examined. Usually, the base overlap is accomplished by positioning polar substituents over the ring system of an adjacent base. The stacking interactions are similar to those found in the crystal structures of other polar aromatic compounds, but are considerably different from the ring-ring interactions in nonpolar aromatic compounds. Apparently, dipole-induced dipole forces are largely responsible for solid-state base stacking. It is found that halogen substituents affect, base-stacking patterns. In general, the presence of a halogen substituent results in a stacking pattern which permits intimate contact between the halogen atom and adjacerit purine or pyrimidine rings. Considering differences in the stacking patterns found for halogenated and nonhalogenated pyrimidines, a niodel is proposed to account for the mutagenic effects of halogenated pyrimidineo.

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Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Human perceptual and cognitive abilities are limited resources. Attention is the mechanism used to allocate such resources in the most effective way. Current technologies, in addition to allowing fast access to information and people, should be designed to support human attentional processes on which they impose further strain. This paper analyses the issues related to the design of systems capable of such support: attention aware systems. We introduce the research aimed at understanding and modelling human attentional processes, including perceptual and cognitive processes as studied in cognitive psychology, as well as rhetorical, aesthetic, and social aspects related to attentional mechanisms. We analyse current approaches to the design of attention aware systems along three major features: detection of user's current attentional state, detection and evaluation of possible alternative attentional states, strategies for focus switch or maintenance. Finally, we discuss the most promising research direction for the development of systems capable of supporting human attentional mechanisms.

N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease. Communication pubs.acs.org/JACS