A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy

Abstract

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients.

RNA Editors, Cofactors, and mRNA Targets: An Overview of the C-to-U RNA Editing Machinery and Its Implication in Human Disease

“…At the same time, it was shown that APOBEC1 is able to edit the GlyR mRNA in vitro. Whereas this is only suggestive (as noted above), recent bioinformatic analyses have identified two genetic variants of APOBEC1, 80M and 80I, which are associated with GlyR editing levels [168]. Further characterization of patients with intractable TLE (iTLE) for APOBEC1 dimorphism, showed that patients with 80I variant suffer from simple or complex seizures, whereas patients with 80M exhibit neurodegeneration and secondary generalized seizure activity [168].…”

Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

“…Investigation of RNA deamination is difficult when compared to similar enzymatic editing activity on other polynucleotides, as there are few systems that can effectively provide a readout for when RNA editing is occurring. A few have been proposed for adenosine deamination [28][29][30], and some have been adapted for cytidine deamination [6,31,32], although these can sometimes be time consuming, show low sensitivity, or are difficult to quantify precisely. Refining on several of these methods led to our development of a system where RNA editing directly induces a shift in subcellular localization of an engineered eGFP [33] construct.…”

APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo

“…We did not consider the role of naturally occurring variants in APOBEC1, for example, which may be a relevant consideration since mutations in APOBEC family genes were shown to modify the editing activity of related hybrid DNA cytosine base editors (Arbab et al 2020). Furthermore, genetic variants of APOBEC1 in humans were associated with altered frequency of GlyR editing (Kankowski et al 2017 (Barker et al 2009). The current findings provide a platform for future approaches to resolve these questions.…”