irCLIP platform for efficient characterization of protein-RNA interactions

Abstract

The complexity of transcriptome-wide protein-RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein-RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein-RNA interactions using far less material than standard protocols.

Figure 1

Ultrasensitive, efficient, and fast CLIP platform facilitates low cell number CLIP, (a) Schematic of irCLIP workflow with improved methodologies versus standard CLIP protocols in italics. Green stars denote internal IR800CW dye and orange ovals 3′ biotin modification. IP, immunoprecipitation. (b–d) Average fold increases in (b) target RNA fragments produced by on-bead versus standard in-lysate nuclease digestions across multiple RBPs; (c) purified RNA resulting from irCLIP reformulation of standard CLIP PK digestions and RNA precipitation; and (d) conversion of purified CLIP RNA to cDNA resulting from irCLIP replacement of the standard CLIP RT. (N = 8 (b), 6 (c), 4 (d) per group.) (e) Approximate time requirement for conversion of precipitated RNA to double-stranded DNA (dsDNA). (f) Approximate required input material for the indicated CLIP methodologies versus irCLIP. (g) Comparison of significant (false discovery rate (FDR) < 0.05) hnRNP-C clusters identified with irCLIP at the CD55 locus using 100,000 or 20,000 cells versus published iCLIP data using approximately 5,000,000 cells. irCLIP-unique clusters are marked above by magenta squares. Green, irCLIP; blue, iCLIP. 11 of 12 irCLIP-unique clusters contained the strong hnRNP-C consensus-binding motif, UUUUU. Three examples are shown to the right. Data plotted as mean ± s.e.m.

Figure 2

irCLIP produces high-quality data sets with greatly reduced cellular inputs, (a) Pearson correlations of RT stops between biological replicates of hnRNP-C irCLIP of the indicated number of cells per immunoprecipitation. (b) Histogram of the unique read fractions of the hnRNP-C irCLIP and iCLIP biological replicates, (c) Top HOMER motif identified for all significant hnRNP-C clusters from the indicated data set. (d) Comparison of significant (FDR < 0.05) hnRNP-C, PTBP1, and HuR clusters identified with irCLIP at the PKM locus. Alt., alternate. Green, irCLIP; blue, iCLIP.