Unbiased identification of trans regulators of ADAR and A-to-I RNA editing

Abstract

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 ...

Genetic control of RNA editing in Neurodegenerative disease

“…The associations between this editing and splicing event may be attributed to the co-regulations of RNA binding proteins, such as DHX9, PRPF8 and U2AF2. Their involvements in alternative splicing and RNA editing have been showed in previous studies (39,60,79,80). According to these analyses, we could further expand the functions of RNA editing QTLs from diverse aspects of phenotype changes.…”

“…First, we assigned edQTLs in different chromosomes, regions, and repeats, to reveal their preferred regulatory distances from corresponding editing events. Second, we identified the edQTLs potentially enriched in RNA binding proteins and their targets from chromatin immunoprecipitation sequencing (CLIPseq) analyses (34)(35)(36)(37)(38), since the previous studies reported their regulatory roles in A-to-I RNA editing (14,39). Third, we recognized the RNA editing QTLs in transcription factors from one previous study (40) or their altered binding motifs discovered by motifbreakR (41), for the further research about the effects of RNA editing QTLs on another phenotype changes.…”

Loss of Cnot6l impairs inosine RNA modifications in mouse oocytes

“…Biological evidence for such a complex is lacking. Recent mass spectrometry approaches to identify ADAR associated proteins that regulate inosine RNA modifications have not identified CCR4-NOT complex components [41]. Rather, several components of the ribosome were identified directly associated with ADAR [41].…”

Learning cis-regulatory principles of ADAR-based RNA editing from CRISPR-mediated mutagenesis

“…Other factors that can be evaluated in the future include the regions in the RNA substrates that may be bound by the dsRNA binding domains (such as illustrated in Fig. 5c) 44 , long-range cis elements such as the editing inducer elements 45 , and trans regulators such as RNA binding proteins 46 . While our current results give rise to models with substantial predictive power for individual substrates, their generalizability remains low (Supplementary Fig.…”