title: Advancing the functional utility of PAR-CLIP by quantifying background binding to mRNAs and lncRNAs.
abstract: BACKGROUND
  Sequence specific RNA binding proteins are important regulators of gene expression. Several related crosslinking-based, high-throughput sequencing methods, including PAR-CLIP, have recently been developed to determine direct binding sites of global protein-RNA interactions. However, no studies have quantitatively addressed the contribution of background binding to datasets produced by these methods.
  
  
  RESULTS
  We measured non-specific RNA background in PAR-CLIP data, demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal among laboratories. We show that quantitative determination of background is essential for identifying targets of most RNA-binding proteins and can substantially improve motif analysis. We also demonstrate that by applying background correction to an RNA binding protein of unknown binding specificity, Caprin1, we can identify a previously unrecognized RNA recognition element not otherwise apparent in a PAR-CLIP study.
  
  
  CONCLUSIONS
  Empirical background measurements of global RNA-protein crosslinking are a necessary addendum to other experimental controls, such as performing replicates, because covalently crosslinked background signals are reproducible and otherwise unavoidable. Recognizing and quantifying the contribution of background extends the utility of PAR-CLIP and can improve mechanistic understanding of protein-RNA specificity, protein-RNA affinity and protein-RNA association dynamics.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Friedersdorf
authors.forename: Jack D
authors.initials: JD
authors.lastname: Keene
mesh_terms: D001665:Binding Sites; D002874:Chromosome Mapping; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D016601:RNA-Binding Proteins