In Vitro Selection with a Site-Specifically Modified RNA Library Reveals the Binding Preferences of N6-Methyladenosine Reader Proteins.
Biochemistry
; 58(31): 3386-3395, 2019 08 06.
Article
em En
| MEDLINE
| ID: mdl-31287290
ABSTRACT
Epitranscriptomic RNA modifications can serve as recognition elements for the recruitment of effector proteins (i.e., "readers") to modified transcripts. While these interactions play an important role in mRNA regulation, there is a major gap in our understanding of the sequence determinants critical for the binding of readers to modified sequence motifs. Here, we develop a high-throughput platform, relying upon in vitro selection with a site-specifically modified random sequence RNA library and next-generation sequencing, to profile the binding specificity of RNA modification reader proteins. We apply our approach to interrogate the effect of sequence context on the interactions of YTH-domain proteins with N6-methyladenosine (m6A)-modified RNA. We find that while the in vitro binding preferences of YTHDC1 strongly overlap with the well-characterized DR(m6A)CH motif, the related YTH-domain proteins YTHDF1 and YTHDF2 can bind tightly to noncanonical m6A-containing sequences. Our results reveal the principles underlying substrate selection by m6A reader proteins and provide a powerful approach for investigating protein-modified RNA interactions in an unbiased manner.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Biblioteca Gênica
/
Adenosina
/
Proteínas de Ligação a RNA
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Estados Unidos