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Updated: May 11, 2026
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
Published on: April 6, 2012
Genome-wide identification of translationally inhibited and degraded miR-155 targets using RNA-interacting protein-IP
Jan Meier1, Volker Hovestadt, Marc Zapatka
1German Cancer Research Center, Division of Molecular Genetics, Heidelberg, Germany.
Abstract:
MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the "RNA-induced silencing complex" (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity.
Insights
This study introduces RNA-interacting protein immunoprecipitation sequencing (RIP-Seq) to precisely identify microRNA targets. The method revealed miR-155 targets involved in cell cycle control, impacting cell proliferation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are crucial regulators of gene expression, influencing cellular functions by targeting messenger RNAs (mRNAs).
- Accurate identification of miRNA targets is essential for understanding their biological roles, but existing methods like miRNA profiling and transcriptome analysis have limitations.
- The RNA-induced silencing complex (RISC), particularly the AGO2 protein, plays a central role in miRNA-mediated gene silencing.
Purpose of the Study:
- To evaluate the precision of RNA-interacting protein immunoprecipitation (RIP) coupled with RNA sequencing (RIP-Seq) for identifying miRNA targets.
- To investigate the specific targets of miR-155 and their functional implications in HEK293T cells.
- To explore potential discrepancies between miRNA expression levels and their actual activity.
Main Methods:
- Utilized AGO2-specific antibodies for immunoprecipitation of RISC-associated mRNAs in HEK293T cells stably expressing miR-155.
- Performed high-throughput RNA sequencing (RIP-Seq) to identify bound mRNAs.
- Integrated RIP-Seq data with in silico predictions and gene expression profiles to analyze miRNA-target interactions and regulatory mechanisms (mRNA decay or translational repression).
Main Results:
- Identified 100 AGO2-associated mRNAs in miR-155-expressing cells, with 67 confirmed as predicted miR-155 targets.
- Demonstrated that identified targets are regulated via mRNA decay or translational repression.
- Found 17 miR-155 targets involved in cell cycle control, correlating with increased cell proliferation observed in miR-155 expressing cells.
- Observed recurrent, disproportionate enrichment of specific miRNAs, including miR-155, in AGO2 precipitates across different cell lines, suggesting potential regulatory complexities.
Conclusions:
- RIP-Seq is a precise and effective method for genome-wide identification of miRNA targets.
- miR-155 directly regulates cell cycle control genes, contributing to enhanced cell proliferation.
- Discrepancies between miRNA expression and AGO2 association suggest complex regulatory mechanisms influencing miRNA activity.
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