Journal Article

Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS)

Pizzinga, Mariavittoria, Harvey, Robert F., Ramakrishna, Manasa, Dezi, Veronica, Elzek, Mohamed, Lilley, Kathryn S., Villanueva, Eneko, Monti, Mie, Queiroz, Rayner M. L., Smith, Tom, Willis, Anne E.

2020

Overview

RNA−protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA−protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein–RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana , Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics. This protocol describes procedures for isolating the protein-bound transcriptome and RNA-binding proteome via UV crosslinking and organic/aqueous phase separation.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/2067 / 631/1647/514/1949 / 631/45/500 / Adducts / Analysis / Analytical Chemistry / Binding / Biological Techniques / Biomedical and Life Sciences / Cell Line / Cell lines / Chemical Fractionation - methods / Computational Biology/Bioinformatics / Crosslinking / E coli / Gene expression / Homeostasis / Humans / Identification and classification / Interfaces / Life Sciences / Materials recovery / Microarrays / Organic Chemistry / Phase separation / Polyadenylation / Protein adducts / Protein binding / Protein interaction / Proteins / Proteome - isolation & purification / Proteome - metabolism / Proteomes / Proteomics / Protocol / Ribonucleic acid / RNA / RNA - isolation & purification / RNA - metabolism / RNA-binding protein / RNA-Binding Proteins - isolation & purification / RNA-Binding Proteins - metabolism / Transcription factors / Transcriptome / Transcriptomes / Workflow