Abstract:
:RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Lambert NJ,Robertson AD,Burge CBdoi
10.1016/bs.mie.2015.02.007subject
Has Abstractpub_date
2015-01-01 00:00:00pages
465-493eissn
0076-6879issn
1557-7988pii
S0076-6879(15)00082-8journal_volume
558pub_type
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