RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins.
Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Nicole
N
Lambert
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Program in Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Alex
A
Robertson
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Mohini
M
Jangi
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Sean
S
McGeary
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Phillip A
PA
Sharp
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: cburge@mit.edu.
Christopher B
CB
Burge
D020816:Amino Acid Motifs; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D000067879:CELF1 Protein; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D051379:Mice; D011485:Protein Binding; D012313:RNA / Q000235:genetics*; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000737:chemistry*; D052156:SELEX Aptamer Technique; D020539:Sequence Analysis, Protein; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity
D000067879:CELF1 Protein; C502743:CELF1 protein, mouse; D004268:DNA-Binding Proteins; C479922:Mbnl1 protein, mouse; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; C558929:Rbfox2 protein, mouse; D012313:RNA