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Identification of microRNA-668 and microRNA-489 targets by RISC immunoprecipitation

ABSTRACT: Identification of microRNA-668 and microRNA-489 targets by RISC immunoprecipitation

PROVIDER: PRJNA483230 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7613130_1.fastq.gz	Fastqsanger.gz
	SRR7613130_2.fastq.gz	Fastqsanger.gz
	SRR7613140_1.fastq.gz	Fastqsanger.gz
	SRR7613140_2.fastq.gz	Fastqsanger.gz
	SRR7613150_1.fastq.gz	Fastqsanger.gz

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Identification of microRNA-668 and microRNA-489 targets by RISC immunoprecipitation

Project description:MicroRNAs inhibits their target mRNA translation by forming RISC complex. Immunoprecipitation of RISC will pull down both microRNA and their target mRNAs. To systemetically examine the potential targets of microRNA-668 and microRNA-489, HEK293 cells were overexpressed with negative control, microRNA-668, or microRNA-489 mimics. Both total RNA samples and RNA samples from RISC complex pulled down by Ago-2 immunoprecipitation were examined by RNA-seq.

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Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.

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