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A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins


Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside–enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link–induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.

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Figure 1: Relationship between the affinity and enrichment of HuR-binding sites obtained with different CLIP methods.
Figure 2: Location of the ten 7-mers with highest affinity for HuR relative to the most frequently cross-linked nucleotide in the binding site.
Figure 3: Predicted affinities of HuR binding sites isolated based on different measures.
Figure 4: Proportion of Ago2 binding sites matching the seed regions of the ten most abundantly expressed miRNA families.
Figure 5: Location and frequency of miRNA seed-complementary regions relative to the most frequently cross-linked nucleotide in the binding site.

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We thank G. Meister (University of Regensburg) for Ago2 antibody, C. Rammelt for initial help with PAR-CLIP, I. Nissen and C. Beisel for support with deep sequencing, J. Krol for help with miRNA quantification, B. Dimitriades for help with cell culture, and W. Keller and W. Filipowicz for critical comments and suggestions. We also thank the members of the Zavolan laboratory for valuable discussions. This work was supported by grants from the Swiss National Science Foundation (#31003A_127307, PDAM_3_127218 and PDFMP3_123123), the Swiss Initiative in Systems Biology (Cell Plasticity) and the Swiss Cancer League (#KFS 02477-08-2009) and by the European Molecular Biology Organization long-term fellowship 184-2009 to S.K.

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S.K. designed and performed the experiments, L.J. designed and performed the experiments and wrote the paper, L.B. analyzed the data, J.H. analyzed the data, M.K. developed the annotation tools and analyzed the data and M.Z. designed and supervised the experiments, analyzed the data and wrote the paper.

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Correspondence to Mihaela Zavolan.

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The authors declare no competing financial interests.

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Kishore, S., Jaskiewicz, L., Burger, L. et al. A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nat Methods 8, 559–564 (2011).

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