Selective Enrichment of A‑to‑I Edited Transcripts from Cellular RNA Using Endonuclease V

Creating accurate maps of A-to-I
RNA editing activity is vital
to improving our understanding of the biological role of this process
and harnessing it as a signal for disease diagnosis. Current RNA sequencing
techniques are susceptible to random sampling limitations due to the
complexity of the transcriptome and require large amounts of RNA material,
specialized instrumentation, and high read counts to accurately interrogate
A-to-I editing sites. To address these challenges, we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving
enzyme, can be repurposed to bind and isolate A-to-I edited transcripts
from cellular RNA. While Mg2+ enables eEndoV to catalyze
RNA cleavage, we show that similar levels of Ca2+ instead
promote binding of inosine without cleavage and thus enable high affinity
capture of inosine in RNA. We leverage this capability to demonstrate
EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing)
as a facile and effective method to enrich A-to-I edited transcripts
prior to RNA-seq, producing significant increases in the coverage
and detection of identified editing sites. We envision the use of
this approach as a straightforward and cost-effective strategy to
improve the epitranscriptomic informational density of RNA samples,
facilitating a deeper understanding of the functional roles of A-to-I
editing.