Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity

• Published in: Cell reports (Cambridge) 2012-05, Vol.1 (5), p.570-581
• Main Authors: Campbell, Zachary T., Bhimsaria, Devesh, Valley, Cary T., Rodriguez-Martinez, Jose A., Menichelli, Elena, Williamson, James R., Ansari, Aseem Z., Wickens, Marvin
• Format: Article
• Language: English
• Subjects: Animals; Caenorhabditis elegans - genetics; Caenorhabditis elegans - metabolism; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Cooperativity; Data processing; High-Throughput Nucleotide Sequencing - methods; Humans; In Vitro Techniques; mRNA; nucleic acids; RNA, Helminth - genetics; RNA, Helminth - metabolism; RNA-binding protein; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sensitivity and Specificity; Two-Hybrid System Techniques
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2012.04.003

The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach, we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules. [Display omitted] ► The SEQRS method is presented for analysis of RNA-protein interactions ► Members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators are analyzed ► Cooperative effects on specificity are triggered by binding of a protein partner ► The specificity of an engineered PUF is described The control and function of RNA are governed by the specificity of proteins to which they bind. Wickens and colleagues describe a method, SEQRS, for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. They identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity.