Selective Enrichment of A-to-I Edited Transcripts from Cellular RNA Using Endonuclease V.
J Am Chem Soc
; 142(11): 5241-5251, 2020 03 18.
Article
en En
| MEDLINE
| ID: mdl-32109061
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Mensajero
/
Edición de ARN
/
Desoxirribonucleasa (Dímero de Pirimidina)
/
Inosina
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
J Am Chem Soc
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos