No abstract

No abstract

Highlights d BioID identifies novel ADAR interactors, including regulators of A-to-I RNA editing d Interactors include all DZF-domain-containing proteins: ILF2, ILF3, STRBP, and ZFR d DZF-domain-containing proteins bind ADAR in an RNAdependent manner d ILF3 is a broadly influential negative regulator of editing

Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.

Highlights d Drosophila Zn72D is a broadly influential regulator of neuronal A-to-I RNA editing d Zn72D regulates ADAR protein levels and editing levels at hundreds of editing sites d Loss of Zn72D causes morphological defects at the neuromuscular junction d Zn72D regulation of editing is conserved in mammalian neurons

15Adenosine-to-inosine RNA editing, catalyzed by ADAR enzymes, alters RNA sequences from 16 those encoded by DNA. These editing events are dynamically regulated, but few trans regulators 17 of ADARs are known in vivo. Here, we screen RNA binding proteins for roles in editing regulation 18 using in vivo knockdown experiments in the Drosophila brain. We identify Zinc-Finger Protein at 19 72D (Zn72D) as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both 20 regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar 21 to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and motility 22 in the fly. Furthermore, the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary 23 neurons, demonstrating the conservation of this regulatory role. The broad and conserved 24 regulation of ADAR editing by Zn72D in neurons represents a novel mechanism by which critically 25 important editing events are sustained. 26Recent studies suggest that regulation of RNA editing levels is highly complex and that critical 53 RNA editing regulators are yet to be identified. RNA editing levels differ across tissues and 54 developmental stages, and these changes do not always correlate with Adar mRNA or protein 55 expression (J. B. Li and Church, 2013;Sapiro et al., 2019; Tan et al., 2017; Wahlstedt et al., 56 2009). Trans regulators of ADAR proteins may help explain this variation in editing levels (Sapiro 57 et al., 2015); however, few ADAR and editing level regulators are known. In mammals, Pin1, 58 WWP2, and AIMP2 regulate ADAR protein levels or localization, which can then alter editing 59 3 levels (Behm et al., 2017; Marcucci et al., 2011; Tan et al., 2017). Editing regulators can also be 60 site-specific, meaning they regulate ADAR editing at only a subset of editing sites rather than 61 globally regulating ADAR activity. Studies in Drosophila identified FMR1 and Maleless as site-62 specific regulators of editing (Bhogal et al., 2011;Reenan et al., 2000). Further study has verified 63 that human homologs of both FMR1 (Tran et al., 2019) and Maleless (Hong et al., 2018a), along 64 with a number of other RNA binding proteins and splicing factors, act as site-specific regulators 65 of RNA editing. These factors, including SRSF9, DDX15, TDP-43, DROSHA, and Ro60 (Garncarz 66 et al., 2013; Quinones-Valdez et al., 2019; Shanmugam et al., 2018; Tariq et al., 2013), help to 67 explain some variation in editing levels; however, with thousands of editing sites in flies and 68 millions in humans (Ramaswami and J. B. Li, 2014), additional regulators likely remain 69 undiscovered. These previous studies highlight RNA binding proteins as strong candidates for 70 editing regulators (Washburn and Hundley, 2016). Because of the conserved roles of Drosophila 71 editing regulators as well as the ability to measure nervous system phenotypes, flies serve as an 72 important model for understanding the regulation of editing as it relates to human...

13Adenosine-to-Inosine RNA editing is catalyzed by ADAR enzymes that deaminate adenosine to 14 inosine. While many RNA editing sites are known, few trans regulators have been identified. We 15 perform BioID followed by mass-spectrometry to identify trans regulators of ADAR1 and ADAR2 16 in HeLa and M17 neuroblastoma cells. We identify known and novel ADAR-interacting proteins. 17Using ENCODE data we validate and characterize a subset of the novel interactors as global or 18 site-specific RNA editing regulators. Our set of novel trans regulators includes all four members 19 of the DZF-domain-containing family of proteins: ILF3, ILF2, STRBP, and ZFR. We show that 20 these proteins interact with each ADAR and modulate RNA editing levels. We find ILF3 is a 21 global negative regulator of editing. This work demonstrates the broad roles RNA binding 22 proteins play in regulating editing levels and establishes DZF-domain containing proteins as a 23 group of highly influential RNA editing regulators. 24 25 47 the majority of ADAR1-regulated editing sites are found in repeat regions, ADAR2 is primarily 48 responsible for editing adenosines found in non-repeat regions, particularly in the brain (Tan et 49 al., 2017). ADAR2-regulated sites in non-repetitive regions include a number of editing events 50 that alter the protein-coding sequences of neuronal RNAs, including GluR2, which encodes a 51 glutamate receptor in which RNA editing is necessary for its proper function. Further 52 demonstrating its important role in neuronal editing, dysregulation of human ADAR2 is 53 associated with multiple neurological diseases, including amyotrophic lateral sclerosis, 54 astrocytoma and transient forebrain ischemia (Slotkin and Nishikura, 2013; Tran et al., 2019). 55Maintaining RNA editing levels is critical for human health, but how RNA editing levels are 56 regulated at specific editing sites across tissues and development is poorly understood. 58While RNA sequence and structure are critical determinants of editing levels, studies querying 59 tissue-specific and developmental-stage-specific editing levels show that the level of editing at 60 the same editing site can vary greatly between individuals and tissues. These changes do not 61 always correlate with ADAR mRNA or protein expression, suggesting a complex regulation of 62 editing events by factors other than ADAR proteins (Sapiro et al., 2019; Tan et al., 2017; 63 Wahlstedt et al., 2009). Recently, an analysis of proteins with an RNA-binding domain profiled 64 by ENCODE suggested that RNA-binding proteins play a role in RNA editing regulation. Further, 65 in mammals, a small number of trans regulators of editing have been identified through 66 functional experiments (Quinones-Valdez et al., 2019). Some of these trans regulators of editing 67 are site-specific, in that they affect editing levels at only a small subset of editing sites. These 68 include RNA binding proteins such as DHX15, HNRNPA2/B1, RPS14, TDP-43, Drosha and 69 Ro60 (Garncarz et al., 2013; Quinones-Valdez ...

Excessive systemic inflammation is characteristic to various acute conditions including sepsis, viral infections and immunotherapy-induced adverse events such as cytokine release syndrome (CRS). Recently, several clinical trials evaluating variants of lipid-formulated RNA vaccines for either cancer or COVID-19 have reported systemic inflammatory responses that limit vaccine dosing in humans. Preclinical studies in inbred laboratory mice failed to predict these adverse events, suggesting the existence of underlying differences in sensitivity to Toll-like receptor (TLR) or other innate agonists between humans and mice. Here, we identify interleukin 1 receptor antagonist (IL-1ra) as an endogenous, inducible suppressor of systemic inflammation. In humans, stimulation with a TLR7/8 adjuvanted RNA-lipoplex (RNA-LPX) vaccine results in the secretion of inflammasome-activated interleukin-1β (IL-1β) by monocytes. Remarkably, IL-1β was found to control the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6) associated with CRS. Unlike humans, murine leukocytes preferentially upregulate anti-inflammatory IL-1ra relative to IL-1β. IL-1ra deletion in mice leads to CRS-like symptoms, indicating that high levels of IL-1ra protect mice from uncontrolled systemic inflammation. Our results demonstrate that IL-1β and IL-1ra are key regulators that control systemic responses to RNA vaccines and other inflammatory stimuli. These data provide an explanation for the dramatic difference in dose-dependent tolerability between mice and humans and suggest an approach to evaluate pathogen-induced or immunotherapy-related inflammatory toxicities in vivo.