TY - JOUR
AU - Lilley, Kathryn
AB - Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein–RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA–protein interactions. We also characterize dynamic changes in RNA–protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.
TI - Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS)
JF - Nature Biotechnology
DO - 10.1038/s41587-018-0001-2
DA - 2019-01-03
UR - https://www.deepdyve.com/lp/springer-journals/comprehensive-identification-of-rna-protein-interactions-in-any-GLCItur740
SP - 169
EP - 178
VL - 37
IS - 2
DP - DeepDyve
ER -