EasyCLIP analysis of RNA-protein interactions incorporating absolute quantification

Douglas F. Porter,Laura K. H. Donohue,Grant A. Goda,Andrew L. Ji,Daniel Dominguez,Maria M. Aleman,Paul A. Khavari,Weili Miao,Xue Yang

doi:10.1038/s41467-021-21623-4

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.

Highlights

Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs
Establishing a distribution for non-RBPs enables the definition of specific target RNAs for any RBP as those interactions with a frequency per protein are unlikely to occur with a randomly selected protein. easyCLIP is applied to the top 33 most frequent missense mutations across 28 RBPs, identifying quantitative changes in RNA binding in specific RBPs that are recurrently mutated in cancer. easyCLIP represents a method with built-in verifications that enables quantification of the number of RNA cross-links per protein in wild-type (WT) and disease-associated mutant RBPs
Two lists of RNA-binding proteins (GO terms and census) were compared with the COSMIC (Catalog of Somatic Mutations in Cancer) list of cancer-associated genes[15] to identify 93 RBPs associated with cancer (Fig. 1a), of which 51 had no clear structured RNAbinding domain (RBD) (Fig. 1b)

Summary

Introduction

Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Proteins important to cancer, such as BRCA1, SMAD3-4, SPEN, CHD2, and JUN, have been categorized as RBPs, yet are not generally studied as such, raising the question as to whether they act in that role Addressing this question for such proteins, and for additional potentially novel RBPs, has been hindered by the lack of a test that quantitates RNA interaction events per protein molecule to provide a global cutoff level of RNA binding to nominate a protein as an RBP. EasyCLIP is applied to the top 33 most frequent missense mutations across 28 RBPs, identifying quantitative changes in RNA binding in specific RBPs that are recurrently mutated in cancer. EasyCLIP represents a method with built-in verifications that enables quantification of the number of RNA cross-links per protein in wild-type (WT) and disease-associated mutant RBPs Establishing a distribution for non-RBPs enables the definition of specific target RNAs for any RBP as those interactions with a frequency per protein are unlikely to occur with a randomly selected protein. easyCLIP is applied to the top 33 most frequent missense mutations across 28 RBPs, identifying quantitative changes in RNA binding in specific RBPs that are recurrently mutated in cancer. easyCLIP represents a method with built-in verifications that enables quantification of the number of RNA cross-links per protein in wild-type (WT) and disease-associated mutant RBPs

Objectives

Methods

Results

Conclusion