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Open Access Highly Accessed Methodology article

Identification of proteins binding coding and non-coding human RNAs using protein microarrays

Zurab Siprashvili1, Dan E Webster1, Markus Kretz1, Danielle Johnston1, John L Rinn2, Howard Y Chang13 and Paul A Khavari14*

Author affiliations

1 The Program in Epithelial Biology, Stanford University School of Medicine, 269 Campus Drive, Room 2145, Stanford, CA 94305, USA

2 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA

3 Howard Hughes Medical Institute, Stanford, CA 94305, USA

4 Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304

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Citation and License

BMC Genomics 2012, 13:633  doi:10.1186/1471-2164-13-633

Published: 16 November 2012

Abstract

Background

The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays.

Results

We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability.

Conclusions

Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.

Keywords:
Non-coding RNA; Microarray; p53; Ras; Staufen