Trans and cis factors affecting A-to-I RNA editing efficiency of a noncoding editing target in C. elegans.

Adenosine-to-inosine RNA editing by ADARs affects thousands of adenosines in an organism's transcriptome. However, adenosines are not edited at equal levels nor do these editing levels correlate well with ADAR expression levels. Therefore, additional mechanisms are utilized by the cell to dictate the editing efficiency at a given adenosine. To examine cis-and trans-acting factors that regulate A-to-I editing levels specifically in neural cells, we utilized the model organism Caenorhabditis elegans We demonstrate that a double-stranded RNA (dsRNA) binding protein, ADR-1, inhibits editing in neurons, which is largely masked when examining editing levels from whole animals. Furthermore, expression of ADR-1 and mRNA expression of the editing target can act synergistically to regulate editing efficiency. In addition, we identify a dsRNA region within the Y75B8A.83' UTR that acts as acis-regulatory element by enhancing ADR-2 editing efficiency. Together, this work identifies mechanisms that regulate editing efficiency of noncoding A-to-I editing sites, which comprise the largest class of ADAR targets.

Controlling the Editor: The Many Roles of RNA-Binding Proteins in Regulating A-to-I RNA Editing.

RNA editing is a cellular process used to expand and diversify the RNA transcripts produced from a generally immutable genome. In animals, the most prevalent type of RNA editing is adenosine (A) to inosine (I) deamination catalyzed by the ADAR family. Throughout development, A-to-I editing levels increase while ADAR expression is constant, suggesting cellular mechanisms to regulate A-to-I editing exist. Furthermore, in several disease states, ADAR expression levels are similar to the normal state, but A-to-I editing levels are altered. Therefore, understanding how these enzymes are regulated in normal tissues and misregulated in disease states is of profound importance. This chapter will both discuss how to identify A-to-I editing sites across the transcriptome and explore the mechanisms that regulate ADAR editing activity, with particular focus on the diverse types of RNA-binding proteins implicated in regulating A-to-I editing in vivo.

Noncoding regions of C. elegans mRNA undergo selective adenosine to inosine deamination and contain a small number of editing sites per transcript.

ADARs (Adenosine deaminases that act on RNA) "edit" RNA by converting adenosines to inosines within double-stranded regions. The primary targets of ADARs are long duplexes present within noncoding regions of mRNAs, such as introns and 3' untranslated regions (UTRs). Because adenosine and inosine have different base-pairing properties, editing within these regions can alter splicing and recognition by small RNAs. However, despite numerous studies identifying multiple editing sites in these genomic regions, little is known about the extent to which editing sites co-occur on individual transcripts or the functional output of these combinatorial editing events. To begin to address these questions, we performed an ultra-deep sequencing analysis of 4 Caenorhabditis elegans 3' UTRs that are known ADAR targets. Synchronous editing events were determined for the long duplexes in vivo. Furthermore, the validity of each editing event was confirmed by sequencing the same regions of mRNA from worms that lack A-to-I editing. This analysis identified a large number of editing sites that can occur within each 3' UTR, but interestingly, each individual transcript contained only a small fraction of these A-to-I editing events. In addition, editing patterns were not random, indicating that an editing event can affect the efficiency of editing at subsequent adenosines. Furthermore, we identified specific sites that can be both positively and negatively correlated with additional sites leading to mutually exclusive editing patterns. These results suggest that editing in noncoding regions is selective and hyper-editing of cellular RNAs is rare.

Development of a Novel Next-Generation Sequencing Assay for Carrier Screening in Old Order Amish and Mennonite Populations of Pennsylvania.

Genetically isolated populations, such as the Old Order Amish and Old Order Mennonite communities, have an increased incidence of specific autosomal recessive disorders caused by the founder effect. In these populations, robust expanded carrier screening and diagnostic testing have the potential to reduce overall medical costs and improve patient outcomes. A novel next-generation sequencing assay was developed using anchored multiplex PCR technology (ArcherDX) for 162 different genetic syndromes caused by 202 pathogenic variants consisting of 150 single-nucleotide changes, 43 small insertion/deletions, and 9 large deletions (>20 nucleotides). To assess the accuracy of the screening panel results, 48 samples were selected on the basis of prior whole exome sequencing results. An additional 15 samples were chosen specifically to validate SMN1 and SMN2 copy number analyses. Collectively, the screening panel detected 273 pathogenic single-nucleotide or small insertion/deletion variants, 35 copy number variations, and 1 chromosomal abnormality (Klinefelter syndrome). Concordance with prior whole exome sequencing was 100%. By using a novel next-generation sequencing workflow, a successful targeted gene variant panel was developed for the Old Order Amish and Old Order Mennonite populations of Lancaster County, Pennsylvania. Population-wide carrier screening may help decrease the morbidity and mortality of these conditions in the high-risk populations.

Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing.

A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes.

The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis.

ADAR proteins alter gene expression both by catalyzing adenosine (A) to inosine (I) RNA editing and binding to regulatory elements in target RNAs. Loss of ADARs affects neuronal function in all animals studied to date. Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis, but the targets responsible for this phenotype remain unknown. To identify critical neural ADAR targets in C. elegans, we performed an unbiased assessment of the effects of ADR-2, the only A-to-I editing enzyme in C. elegans, on the neural transcriptome. Development and implementation of publicly available software, SAILOR, identified 7361 A-to-I editing events across the neural transcriptome. Intersecting the neural editome with adr-2 associated gene expression changes, revealed an edited mRNA, clec-41, whose neural expression is dependent on deamination. Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect, providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression.

The dsRBP and inactive editor ADR-1 utilizes dsRNA binding to regulate A-to-I RNA editing across the C. elegans transcriptome.

Inadequate adenosine-to-inosine editing of noncoding regions occurs in disease but is often uncorrelated with ADAR levels, underscoring the need to study deaminase-independent control of editing. C. elegans have two ADAR proteins, ADR-2 and the theoretically catalytically inactive ADR-1. Using high-throughput RNA sequencing of wild-type and adr mutant worms, we expand the repertoire of C. elegans edited transcripts over 5-fold and confirm that ADR-2 is the only active deaminase in vivo. Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3' UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2, and mutations within its double-stranded RNA (dsRNA) binding domains abolish both binding and editing regulation. We conclude that ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo. These results raise the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing through deaminase-independent mechanisms.