Zinc finger RNA binding protein Zn72D regulates ADAR-mediated RNA editing in neurons

Abstract

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...

ExOrthist: a tool to infer exon orthologies at any evolutionary distance

“…We used ExOrthist to compare sets of exons regulated by the splicing factor Nova in mouse and fruitfly. For this purpose, we ran vast-tools [27] to obtain changes in inclusion levels (ΔPSIs) for all exons upon Nova2 depletion in mouse embryonic cortex [39] and upon knockdown of the fruifly ortholog ( ps ) in whole adult brains [40]; we then defined as Nova -dependent exons in each species those with a |ΔPSI| > 15 (827 in mouse and 407 in fruifly, respectively; Fig. 5B).…”

“…To identify Nova -dependent exons, we run vast-tools v2.5.1 for two datasets characterized by deficiency of Nova orthologs in each species. In particular, we used an experiment with Nova2 depletion in mouse embryonic cortex at the E18.5 stage [39] and another one with downregulation of pasilla ( ps ) in adult fly whole brains [40]. To obtain inclusion levels for all exons, we employed vast-tools align and combine with default parameters for mm10 and dm6 (VASTDB libraries: vastdb.mm2.23.06.20.tar.gz and vastdb.dme.23.06.20.tar.gz) [27].…”

The Integrative Studies on the Functional A-to-I RNA Editing Events in Human Cancers

“…It revealed the possible multi-regulators of A-to-I RNA editing. From previous literature, these diverse regulatory mechanisms probably include genetic variations (67,68), splicing efficiency (69,70), and RNA binding proteins (71,72). For a functional RNA editing candidate (CAediting_1478179 of APOL1) proposed in this study, we also discovered its differential editing frequencies among the genotyping groups of three single nucleotide variations in the KIRC cancer type (Fig.…”

RNA editing‐mediated regulation of calcium‐dependent activator protein for secretion (CAPS1) localization and its impact on synaptic transmission

“…Alternatively, as suggested by differences in effect size, additional regulatory factors may be involved. Numerous studies spanning the past decade have consistently concluded that changes in ADAR expression do not fully account for differences in the extent of A‐to‐I editing and the identification of such regulatory factors remains an active area of research for the field (Hood et al., 2014; Li & Church, 2013; Porath et al., 2019; Sapiro et al., 2020; Schaffer et al., 2020; Tan et al., 2017; Wahlstedt et al., 2009).…”