Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition.

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.

The Effect of Tactical Tasks and Gear on Muscle Activation of SWAT Officers.

ABSTRACT: Keeler, JM, Pohl, MB, Bergstrom, HC, Thomas, JM, and Abel, MG. The effect of tactical tasks and gear on muscle activation of SWAT officers. J Strength Cond Res 36(1): 238-244, 2022-Special Weapons and Tactics (SWAT) officers perform a variety of tactical operations while wearing tactical gear. Load carriage has been shown to alter muscle activation in the torso and is also associated with lower back pain, which is a prevalent musculoskeletal injury suffered by SWAT Officers. The purpose of this study was to quantify the effect of tactical gear on muscle activation of torso musculature while performing occupational tasks. Twenty male SWAT Officers (age: 34.7 ± 4.5 years; height: 1.79 ± 0.1 m; body mass: 91.5 ± 17.3 kg) performed 4 tasks (standing, rifle walk, sitting, and shield walk) with and without gear (mass of gear: 13.8 ± 1.9 kg). Mean electromyographic amplitude was evaluated bilaterally for the erector spinae, rectus abdominis, and external oblique muscles during the trials and expressed relative to maximal voluntary isometric contraction (MVIC). Addition of gear significantly increased erector spinae mean muscle activation during the rifle walk task (mean delta: +0.16%). However, no differences in muscle activation were identified for any other muscles between gear conditions (effect size ≤ 0.15). The shield walk produced the highest mean activation for each muscle during different tasks. The dynamic tasks yielded (0.24-4.18% MVIC) greater muscle activation levels than sitting and standing tasks. Despite minimal increases in muscle activation levels with the addition of gear, load carriage is known to increase the risk of acute and chronic injury. Collectively, these findings indicate that SWAT Officers should perform most skills without gear during tactical training to simulate task-specific movement patterns but reduce the risk of musculoskeletal injury.

How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs.

Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.

A Chemical Signature for Cytidine Acetylation in RNA.

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.

Synthesis of native-like crosslinked duplex RNA and study of its properties.

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.

Sepsis as the Grand Mimic of Secondary Hemophagocytic Lymphohistiocytosis: Serratia marcescens Bacteremia with Concomitant Decompensated Cirrhotic Liver Disease.

Secondary hemophagocytic lymphohistiocytosis (HLH) is an elusive entity with sequelae that may be confused with sepsis. We discuss a 45-year-old man with decompensated liver cirrhosis with sepsis treated with broad-spectrum intravenous antibiotics. Further work-up initially supported sepsis-HLH overlap syndrome (SHLHOS) and corticosteroids were added. Ongoing refractory hypotension ensued, and the patient passed within 31 hours of presentation. Based on the patient's overwhelming immune activation and clinical course likely unsalvageable by cytotoxic immunosuppressive agents, the patient was diagnosed with sepsis with acute end organ dysfunction. This case report illustrates both the diagnostic challenge of sepsis versus HLH, which both require very different treatments, and the potential for rapid clinical decline without swift recognition and management of the true pathology.

Remodelin Is a Cryptic Assay Interference Chemotype That Does Not Inhibit NAT10-Dependent Cytidine Acetylation.

Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in ALARM NMR and proteome-wide affinity profiling assays. Biophysical analyses find no direct evidence for interaction of remodelin with the NAT10 acetyltransferase active site. Cellular studies verify that N4-acetylcytidine (ac4C) is a nonredundant target of NAT10 activity in human cell lines and find that this RNA modification is not affected by remodelin treatment in several orthogonal assays. These studies display the potential for remodelin's chemotype to interact with multiple protein targets in cells and indicate remodelin should not be applied as a specific chemical inhibitor of NAT10-catalyzed RNA acetylation.

Metabolic Regulation of the Epitranscriptome.

An emergent theme in cancer biology is that dysregulated energy metabolism may directly influence oncogenic gene expression. This is due to the fact that many enzymes involved in gene regulation use cofactors derived from primary metabolism, including acetyl-CoA,  S-adenosylmethionine, and 2-ketoglutarate. While this phenomenon was first studied through the prism of histone and DNA modifications (the epigenome), recent work indicates metabolism can also impact gene regulation by disrupting the balance of RNA post-transcriptional modifications (the epitranscriptome). Here we review recent studies that explore how metabolic regulation of writers and erasers of the epitranscriptome (FTO, TET2, NAT10, MTO1, and METTL16) helps shape gene expression through three distinct mechanisms: cofactor inhibition, cofactor depletion, and writer localization. Our brief survey underscores similarities and differences between the metabolic regulation of the epigenome and epitranscriptome, and highlights fertile ground for future investigation.

Nucleotide resolution sequencing of N4-acetylcytidine in RNA.

Posttranscriptional modifications of RNA represent an emerging class of regulatory elements in human biology. Improved methods for studying how these elements are controlled and where they occur has the potential to transform our understanding of gene expression in development and disease. Here we describe a chemical method for nucleotide resolution sequencing of N4-acetylcytidine (ac4C), a highly conserved modified nucleobase whose formation is catalyzed by the essential cytidine acetyltransferase enzyme NAT10. This approach enables the sensitive, PCR-amplifiable detection of individual ac4C sites from nanograms of unfractionated cellular RNA. The sensitive and quantitative nature of this assay provides a powerful tool to understand how cytidine acetylation is targeted, profile RNA acetyltransferase dynamics, and validate the sites and stoichiometry of ac4C in novel RNA species.

Insulin-like growth factor (IGF)-II- mediated fibrosis in pathogenic lung conditions.

Type 2 insulin-like growth factor (IGF-II) levels are increased in fibrosing lung diseases such as idiopathic pulmonary fibrosis (IPF) and scleroderma/systemic sclerosis-associated pulmonary fibrosis (SSc). Our goal was to investigate the contribution of IGF receptors to IGF-II-mediated fibrosis in these diseases and identify other potential mechanisms key to the fibrotic process. Cognate receptor gene and protein expression were analyzed with qRT-PCR and immunoblot in primary fibroblasts derived from lung tissues of normal donors (NL) and patients with IPF or SSc. Compared to NL, steady-state receptor gene expression was decreased in SSc but not in IPF. IGF-II stimulation differentially decreased receptor mRNA and protein levels in NL, IPF, and SSc fibroblasts. Neutralizing antibody, siRNA, and receptor inhibition targeting endogenous IGF-II and its primary receptors, type 1 IGF receptor (IGF1R), IGF2R, and insulin receptor (IR) resulted in loss of the IGF-II response. IGF-II tipped the TIMP:MMP balance, promoting a fibrotic environment both intracellularly and extracellularly. Differentiation of fibroblasts into myofibroblasts by IGF-II was blocked with a TGFß1 receptor inhibitor. IGF-II also increased TGFß2 and TGFß3 expression, with subsequent activation of canonical SMAD2/3 signaling. Therefore, IGF-II promoted fibrosis through IGF1R, IR, and IGF1R/IR, differentiated fibroblasts into myofibroblasts, decreased protease production and extracellular matrix degradation, and stimulated expression of two TGFß isoforms, suggesting that IGF-II exerts pro-fibrotic effects via multiple mechanisms.

A Bump-Hole Approach for Directed RNA Editing.

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity.

Profiling Cytidine Acetylation with Specific Affinity and Reactivity.

The human acetyltransferase NAT10 has recently been shown to catalyze formation of N4-acetylcytidine (ac4C), a minor nucleobase known to alter RNA structure and function. In order to better understand the role of RNA acetyltransferases in biology and disease, here we report the development and application of chemical methods to study ac4C. First, we demonstrate that ac4C can be conjugated to carrier proteins using optimized protocols. Next, we describe methods to access ac4C-containing RNAs, enabling the screening of anti-ac4C antibodies. Finally, we validate the specificity of an optimized ac4C affinity reagent in the context of cellular RNA by demonstrating its ability to accurately report on chemical deacetylation of ac4C. Overall, these studies provide a powerful new tool for studying ac4C in biological contexts, as well as new insights into the stability and half-life of this highly conserved RNA modification. More broadly, they demonstrate how chemical reactivity may be exploited to aid the development and validation of nucleobase-targeting affinity reagents designed to target the emerging epitranscriptome.

Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity.

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the 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 human ADAR2 deaminase domain 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 of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus 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.

Transciliary supraorbital approach (eyebrow approach) for resection of retrochiasmatic craniopharyngiomas: an alternative approach, case series, and literature review.

INTRODUCTION: Craniopharyngiomas are known for an irregular growth pattern and extension into the retrochiasmatic space with adherence to adjacent structures. We describe the use of the transciliary supraorbital approach (eyebrow approach) for resection of craniopharyngiomas with retrochiasmatic extension. METHODS: Our clinical database was reviewed to identify operative cases of craniopharyngiomas between July 1998 and January 2011. Only patients who had retrochiasmatic extension and underwent an eyebrow approach were included in this analysis. Endoscopy was used to aid surgical resection at the discretion of the surgeon. Six patients were identified (three from a retrospective review and three during the course of prospective follow-up evaluation). RESULTS: The group included three men and three women (mean age: 41.2 years; range: 28-57 years). All patients had visual and hormonal deficits at presentation. Complete resection was achieved in four patients. Endoscopic assistance was used in three patients. Opening of the frontal sinus occurred in two cases during surgery; an additional surgical procedure was required for repair of a cerebrospinal fluid leak in one of these cases. The duration of clinical follow-up ranged from 18 to 97 months. All patients had excellent cosmetic results, visual improvement to a serviceable level, and a Karnofsky performance scale score ≥ 80. CONCLUSIONS: The eyebrow approach is safe for resection of craniopharyngiomas with retrochiasmatic extension. This approach offers a reasonable surgical corridor for resection of the retrochiasmatic tumor component and can be enhanced using endoscopic assistance.

Malignant pleural effusions: a review.

Malignant pleural effusions are a cause of significant symptoms and distress in patients with end-stage malignancies and portend a poor prognosis. Management is aimed at symptom relief, with minimally invasive interventions and minimal requirement for hospital length of stay. The management options include watchful waiting if no symptoms are present, repeat thoracentesis, medical or surgical thoracoscopic techniques to achieve pleurodesis, pleuroperitoneal shunts, placement of tunneled pleural catheters, or a combination of modalities. To determine the best modality for management, patients must be assessed individually with concern for symptoms, functional status, prognosis, and their social and financial situations.

Modifiers of symptomatic embolic risk in infective endocarditis.

OBJECTIVE: To ascertain the impact of prior antiplatelet and statin therapy on symptomatic embolic events in [corrected] infective endocarditis (IE). PATIENTS AND METHODS: We studied a retrospective cohort of adult patients with a diagnosis of IE who presented to Mayo Clinic (Rochester, MN) from January 1, 2003, to December 31, 2006. Patients were grouped into those who received treatment before infection or controls who did not receive treatment for both antiplatelet therapy and, separately, statin therapy. Because of the retrospective study design and thus the nonrandomized treatment groups, a propensity score approach was used to account for the confounding factors that may have influenced treatment allocation. Antiplatelet therapy included aspirin, dipyridamole, clopidogrel, ticlopidine or any combination of these agents. Statin therapy included atorvastatin, simvastatin, pravastatin, lovastatin, rosuvastatin, or fluvastatin. The primary end point was a symptomatic embolic event that occurred before or during hospitalization. Multivariable logistic regression was used to assess the propensity-adjusted effects of continuous daily therapy with antiplatelet and statin agents on risk of symptomatic emboli. Likewise, Cox proportional hazards regression was used to test for an independent association with 6-month mortality for each of the treatments. RESULTS: The study cohort comprised 283 patients with [corrected] IE. Twenty-eight patients (24.1%) who received prior continuous antiplatelet therapy developed a symptomatic embolic event compared with 66 (39.5%) who did not receive such treatment. After adjusting for propensity to treat, the effect of antiplatelet therapy on embolic risk was not statistically significant (odds ratio, 0.71; 95% confidence interval [CI], 0.37-1.36; P=.30). Only 14 patients (18.2%) who received prior continuous statin therapy developed a symptomatic embolic event compared with 80 (39.4%) of the 203 patients who did not. After adjusting for propensity to treat with statin therapy, the benefit attributable to statins was significant (odds ratio, 0.30; 95% CI, 0.14-0.62; P=.001). The 6-month mortality rate of the entire cohort was 28% (95% CI, 23%-34%). No significant difference was found in the propensity-adjusted rate of 6-month mortality between patients who had and had not undergone prior antiplatelet therapy (P=.91) or those who had and had not undergone prior statin therapy (P=.87). CONCLUSION: The rate of symptomatic emboli associated with IE was reduced in patients who received continuous daily statin therapy before onset of IE. Despite fewer embolic events observed in patients who received antiplatelet agents, a significant association was not found after adjusting for propensity factors. A continued evaluation of these drugs and their potential impact on subsequent embolism among IE patients is warranted.

Epidemiological trends of infective endocarditis: a population-based study in Olmsted County, Minnesota.

OBJECTIVE: To provide a contemporary profile of epidemiological trends of infective endocarditis (IE) in Olmsted County, Minnesota. PATIENTS AND METHODS: This study consists of all definite or possible IE cases among adults in Olmsted County from January 1, 1970, through December 31, 2006. Cases were identified using resources of the Rochester Epidemiology Project. RESULTS: We identified 150 cases of IE. The age- and sex-adjusted incidences of IE ranged from 5.0 to 7.9 cases per 100,000 person-years with an increasing trend over time differential with respect to sex (for interaction, P=.02); the age-adjusted incidence of IE increased significantly in women (P=.006) but not in men (P=.79). We observed an increasing temporal trend in the mean age at diagnosis (P=.04) and a decreasing trend in the proportion of cases with rheumatic heart disease as a predisposing condition (P=.02). There were no statistically significant temporal trends in the incidence of either Staphylococcus aureus or viridans group streptococcal IE. Data on infection site of acquisition were available for cases seen in 2001 and thereafter, with 50.0% designated as health care-associated, 42.5% community-acquired, and 7.5% nosocomial. CONCLUSION: The incidence of IE among women increased from 1970 to 2006. Ongoing surveillance is warranted to determine whether the incidence change in women will be sustained. Subsequent analysis of infection site of acquisition and its impact on the epidemiology of IE are planned.

AMPA receptor downscaling at the onset of Alzheimer's disease pathology in double knockin mice.

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-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) contributes to pathology in double knockin (2 x KI) mice carrying human mutations in the genes for amyloid precursor protein and presenilin-1. By using whole-cell recordings, we show that 2 x KI mice exhibit age-related downscaling of AMPAR-mediated evoked currents and spontaneous, miniature currents. Electron microscopic analysis further corroborates the synaptic AMPAR decrease. Additionally, 2 x KI mice show age-related deficits in bidirectional plasticity (long-term potentiation and long-term 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.