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

Abstract

Summary 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.

Programmable RNA-binding protein composed of repeats of a single modular unit

“…Along these lines, other members of the Puf family have also been used to engineer selective binding between functional effector proteins and RNA targets. One of the most extensively studied is the C. elegans Fem-3 mRNA-binding factor 2 (FBF-2), which is an analog of PumHD (6,(37)(38)(39)(40). The seemingly simple modular binding nature of PumHD masks a great wealth of complexity in the way that the diverse units of the protein contribute to overall protein binding.…”

RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

“…Aliquots of protein were stored in 1ϫ SEQRS buffer (50 mM Tris-HCl, pH 8, 0.1 mM MgCl 2 , 150 mM NaCl, 0.1% Nonidet P-40) containing 20% glycerol prior to flash freezing and storage at Ϫ80°C. The SEQRS experiment was performed as described with minor modifications (47). Briefly, the initial libraries were transcribed from 1 g of input dsDNA using the AmpliScribe T7-flash transcription kit (Epicenter).…”

“…To further characterize the binding specificity of STAU-1, we used a method termed SEQRS (in vitro selection, high throughput sequencing of RNA) (47). In this method, reiterated rounds of selection from RNA pools are followed by deep sequencing.…”

“…The number of reads obtained correlates with independent measures of binding affinity (i.e. electrophoretic mobility shift and yeast three-hybrid assays) (47).…”

“…E, analysis of STAU-1 specificity for RNA structure in vitro. The binding of full-length STAU-1 to double-stranded RNAs of variable sequence was determined following five rounds of selection using the SEQRS method (47). In these experiments, the number of reads correlates with binding affinity.…”