@article{389d8c85532b4d48b29bb54c13077f0a,
title = "easyCLIP analysis of RNA-protein interactions incorporating absolute quantification",
abstract = "Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.",
author = "Porter, {Douglas F.} and Weili Miao and Xue Yang and Goda, {Grant A.} and Ji, {Andrew L.} and Donohue, {Laura K.H.} and Aleman, {Maria M.} and Daniel Dominguez and Khavari, {Paul A.}",
note = "Funding Information: We thank Brian Zarnegar for reagents, and input on experiments and their interpretation. We also thank Amin Zia, Zurab Siprashvili, and Yuning Wei for assistance. SF3B1 was obtained from a vector produced by Angelos Constantinou, provided by Marc-Henri Stern. Funding was provided by a USVA Merit Review grant BX001409 to P.A.K. and by NIAMS/NIH grants AR49737 and AR45192 to P.A.K., and 1F32AR072504 to D.F.P.; A.L.J. is a recipient of a Physician-Scientist Training Award from the Damon Runyon Cancer Research Foundation. Some data were generated on an Illumina HiSeq purchased with funds from award S10OD018220 by SFGF at Stanford. High-throughput sequencing data are given under the accessions GSE154168, GSE162366, and GSE131210. Publisher Copyright: {\textcopyright} 2021, This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.",
year = "2021",
month = dec,
day = "1",
doi = "10.1038/s41467-021-21623-4",
language = "English",
volume = "12",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Publishing Group",
number = "1",
}