Transcriptomics

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RNAcompete: a method for rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins


ABSTRACT: Metazoan genomes encode hundreds of RNA binding proteins (RBPs) but relatively few have well-defined RNA-binding preferences. Current techniques for determining RNA targets, including those involving in vitro selection and RNA co-immunoprecipitation, require significant time and labour investment. Here we introduce RNAcompete, a new method for the systematic analysis of RNA-binding specificities that employs a single binding reaction to determine the relative preferences of RBPs for short RNAs that containing a complete range of k-mers in structured and unstructured RNA contexts. We tested RNAcompete by analyzing nine diverse RBPs (HuR, Vts1, FUSIP1, PTB, U1A, SF2/ASF, SLM2, RBM4, and YB1). RNAcompete identified both expected and previously unknown RNA binding preferences. Using in vitro and in vivo binding data, we demonstrate that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than conventional motif models. We anticipate that RNAcompete will be a valuable tool for the large-scale study of RNA-protein interactions. The bound RNA from each RNA binding protein pulldown assay is analyzed on a custom Agilent microarray using a pool RNA control as a reference.

ORGANISM(S): synthetic construct  

SUBMITTER: Debashish Ray   Esther T Chan  Benjamin J Blencowe  Timothy R Hughes  Quaid D Morris  Shaheynoor Talukder  Hilal Kazan  Lourdes Pena-Castillo  Sidharth Chaudhry 

PROVIDER: E-GEOD-15769 | ArrayExpress | 2010-05-18

SECONDARY ACCESSION(S): GSE15769PRJNA116807

REPOSITORIES: GEO, ArrayExpress

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Publications

Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins.

Ray Debashish D   Kazan Hilal H   Chan Esther T ET   Peña Castillo Lourdes L   Chaudhry Sidharth S   Talukder Shaheynoor S   Blencowe Benjamin J BJ   Morris Quaid Q   Hughes Timothy R TR  

Nature biotechnology 20090628 7


Metazoan genomes encode hundreds of RNA-binding proteins (RBPs) but RNA-binding preferences for relatively few RBPs have been well defined. Current techniques for determining RNA targets, including in vitro selection and RNA co-immunoprecipitation, require significant time and labor investment. Here we introduce RNAcompete, a method for the systematic analysis of RNA binding specificities that uses a single binding reaction to determine the relative preferences of RBPs for short RNAs that contai  ...[more]

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