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PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
Published on: July 2, 2010
Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps
Sung Wook Chi1, Julie B Zang, Aldo Mele
1Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Nature
|June 19, 2009
Summary
High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) identified microRNA (miRNA) and target messenger RNA (mRNA) interactions in the mouse brain. This method maps miRNA binding sites, advancing understanding of gene regulation.
Area of Science:
- Molecular Biology
- Genomics
- Neuroscience
Background:
- MicroRNAs (miRNAs) regulate gene expression through base pairing with messenger RNAs (mRNAs).
- Predicting miRNA-mRNA interactions is challenging due to the short sequence (6-8 nucleotides) required for binding.
- High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) identifies functional protein-RNA interactions.
Purpose of the Study:
- To use HITS-CLIP to map miRNA and mRNA binding sites in vivo.
- To identify specific miRNA-mRNA interactions in the mouse brain.
- To provide a platform for exploring miRNA action and targeting clinically relevant interactions.
Main Methods:
- Covalent crosslinking of native argonaute (Ago) protein-RNA complexes in mouse brain.
- Utilizing HITS-CLIP to generate simultaneous Ago-miRNA and Ago-mRNA binding site data.
- Bioinformatic analysis to identify miRNA-mRNA interaction sites.
- Genome-wide validation of interaction maps for miR-124 and mapping of 20 abundant miRNAs.
Main Results:
- Generated simultaneous Ago-miRNA and Ago-mRNA binding site data from mouse brain.
- Identified genome-wide interaction maps for miR-124.
- Created interaction maps for the 20 most abundant miRNAs in P13 mouse brain.
- Established precise sequences for miRNA-mRNA targeting.
Conclusions:
- Ago HITS-CLIP is a versatile platform for studying miRNA specificity and function in vivo.
- This approach enables the identification of precise miRNA-mRNA interactions.
- The findings facilitate the targeting of clinically relevant miRNA-mRNA interactions.
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