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

Abstract

Metazoan genomes encode hundreds of RNA-binding proteins (RBPs) but RNA-binding preferences for relatively few RBPs have been well defined1. Current techniques for determining RNA targets, including in vitro selection and RNA co-immunoprecipitation2,3,4,5, 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 contain 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 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 are conventional motif models. We anticipate that RNAcompete will be a valuable tool for the study of RNA-protein interactions.

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Figure 1: The RNAcompete method and example data for HuR and Vts1.
Figure 2: RNAcompete analysis of nine different RBPs, representing four different classes of RNA binding domains.
Figure 3: ROC curves showing the ability of different representations of RNA-binding activity to predict bound versus unbound sequences in vivo.

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Acknowledgements

We are grateful to C. Smibert, H. Lipshitz, F. Sicheri, M. Kekis and T. Babak for helpful commentary. Bacterial expression plasmids for the N-terminal arm of U1A, the Vts1 SAM domain, full-length PTB and YB1 were generously provided by C. Lutz (Univ. of Medicine and Dentistry of New Jersey), C. Smibert (Univ. of Toronto), M. Garcia-Blanco (Duke Univ.) and K. Kohno (Univ. of Occupational and Environmental Health, Kitakyushu, Japan). This work was supported by grants to T.R.H., B.J.B. and Q.M. from CIHR (MOP-49451, MOP-14609, MOP-93671), by Natural Sciences and Engineering Research Council operating and Canadian Foundation of Innovation grants to Q.M., Genome Canada through the Ontario Genomics Institute and the Ontario Research Fund. D.R. was supported in part by a National Science and Engineering Research Council of Canada (NSERC) postdoctoral fellowship.

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D.R. developed the method and performed the experiments; D.R., H.K., Q.M. and T.R.H. designed the array, processed and analyzed the data, wrote the paper and made the figures; E.C. and L.P.C. contributed to the motif analyses and cross-validation; S.C. and S.T. assisted with cloning and protein production; B.J.B., Q.M. and T.R.H. conceived of the method and supported the project.

Corresponding authors

Correspondence to Quaid Morris or Timothy R Hughes.

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Supplementary Figures 1–5, Supplementary Table 1 (PDF 394 kb)

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Ray, D., Kazan, H., Chan, E. et al. Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins. Nat Biotechnol 27, 667–670 (2009). https://doi.org/10.1038/nbt.1550

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