a6A-seq: N 6-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing.
Fundam Res
; 3(5): 657-664, 2023 Sep.
Article
en En
| MEDLINE
| ID: mdl-38933292
ABSTRACT
The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics. The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs. Here we developed an a6A-seq method, which takes advantage of N6-allyladenosine (a6A) metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner. a6A plays a role as a chemical sequencing tag in that the iodination of a6A in mRNAs results in 1,N 6-cyclized adenosine (cyc-A), which induces base misincorporation during RNA reverse transcription, thus making a6A-labelled mRNAs detectable by sequencing. A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine. a6A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition. Compared with regular RNA-seq, a6A-seq could more sensitively detect the change of mRNA production over a time scale. The experiment of a6A-containing mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a6A-seq data. Together, our results show a6A-seq is an effective tool to study RNA dynamics.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Fundam Res
Año:
2023
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
China