RNA Sequence Context Effects Measured In Vitro Predict In Vivo Protein Binding and Regulation

• Published in: Molecular cell 2016-10, Vol.64 (2), p.294-306
• Main Authors: Taliaferro, J. Matthew, Lambert, Nicole J., Sudmant, Peter H., Dominguez, Daniel, Merkin, Jason J., Alexis, Maria S., Bazile, Cassandra A., Burge, Christopher B.
• Format: Article
• Language: English
• Subjects: Algorithms; Alternative Splicing; Animals; Binding Sites; Cattle; Cell Differentiation; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fibroblasts - cytology; Fibroblasts - metabolism; Gene Expression; Macaca; Mice; Mice, Knockout; Mouse Embryonic Stem Cells - cytology; Mouse Embryonic Stem Cells - metabolism; mutagenesis; Mutation; Neurons - cytology; Neurons - metabolism; Nucleic Acid Conformation; Nucleotide Motifs; nucleotide sequences; post-transcriptional; Protein Binding; Protein Interaction Domains and Motifs; Rats; RBNS; RNA; RNA - chemistry; RNA - genetics; RNA - metabolism; RNA processing; RNA Splicing Factors - chemistry; RNA Splicing Factors - genetics; RNA Splicing Factors - metabolism; RNA-binding proteins; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Software
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2016.08.035

Many RNA binding proteins (RBPs) bind specific RNA sequence motifs, but only a small fraction (∼15%–40%) of RBP motif occurrences are occupied in vivo. To determine which contextual features discriminate between bound and unbound motifs, we performed an in vitro binding assay using 12,000 mouse RNA sequences with the RBPs MBNL1 and RBFOX2. Surprisingly, the strength of binding to motif occurrences in vitro was significantly correlated with in vivo binding, developmental regulation, and evolutionary age of alternative splicing. Multiple lines of evidence indicate that the primary context effect that affects binding in vitro and in vivo is RNA secondary structure. Large-scale combinatorial mutagenesis of unfavorable sequence contexts revealed a consistent pattern whereby mutations that increased motif accessibility improved protein binding and regulatory activity. Our results indicate widespread inhibition of motif binding by local RNA secondary structure and suggest that mutations that alter sequence context commonly affect RBP binding and regulation. [Display omitted] •Most high-affinity RBP motifs in expressed RNAs are not bound in vivo•A high-throughput in vitro interaction assay discriminates in vivo-bound motifs•Motifs near ancient exons have reduced RNA structure and stronger RBP binding•Deep mutagenesis reveals local RNA structure as a key binding determinant Taliaferro et al. use high-throughput in vitro binding assays and deep mutational screening to reveal that local RNA structure is a key determinant of in vitro RBP-RNA interaction and predictive of in vivo binding and regulation.