RISC RNA sequencing for context-specific identification of in vivo microRNA targets.
Circ Res
; 108(1): 18-26, 2011 Jan 07.
Article
en En
| MEDLINE
| ID: mdl-21030712
ABSTRACT
RATIONALE MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. OBJECTIVE:
To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). METHODS ANDRESULTS:
We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts.CONCLUSIONS:
RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN Mensajero
/
Análisis de Secuencia de ARN
/
Perfilación de la Expresión Génica
/
Complejo Silenciador Inducido por ARN
/
MicroARNs
/
Miocardio
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Revista:
Circ Res
Año:
2011
Tipo del documento:
Article
País de afiliación:
Estados Unidos