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. 2017 Apr 15:118-119:41-49.
doi: 10.1016/j.ymeth.2016.11.009. Epub 2016 Nov 18.

PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites

Daniel Benhalevy  1 , Hannah L McFarland  1 , Aishe A Sarshad  1 , Markus Hafner  2
Affiliations

PAR-CLIP and streamlined small RNA cDNA library preparation protocol for the identification of RNA binding protein target sites

Daniel Benhalevy et al. Methods. .

Abstract

The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale. PAR-CLIP involves irreversible UV-mediated crosslinking of RNAs labeled with photoreactive nucleosides to interacting proteins, followed by stringent purification steps and the conversion of crosslinked RNA into small RNA cDNA libraries compatible with next-generation sequencing. The defining hallmark of PAR-CLIP is a diagnostic mutation at the crosslinking site that is introduced into cDNA during the library preparation process. This feature allows for efficient computational removal of contaminating sequences derived from non-crosslinked fragments of abundant cellular RNAs. In the following, we present two different step-by-step procedures for PAR-CLIP, which differ in the small RNA cDNA library preparation procedure: (1) Standard library preparation involving gel size selections after each enzymatic manipulation, and (2) A modified PAR-CLIP procedure ("on-beads" PAR-CLIP), where most RNA manipulations including the necessary adapter ligation steps are performed on the immobilized RNP. This streamlined procedure reduces the protocol preparation time by three days compared to the standard workflow.

Keywords: Next-generation sequencing; PAR-CLIP; Posttranscriptional Gene Regulation; RNA binding protein; Small RNA cDNA library.

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Figures

Fig. 1.
Fig. 1.
Overview of key steps in PAR-CLIP for the standard (left panel) and “on beads” (right panel) approaches. These methods differ in the small RNA cDNA library preparation steps, which are shortened in the “on beads” protocol by three days. In the latter approach, all adapter ligation steps are performed on matrix-immobilized RNPs, thus avoiding gel fractionation steps.
Fig. 2.
Fig. 2.
Illustration of steps during PAR-CLIP. (A) Immunoblot using antibody against the YBX1 protein. Samples from left to right: input, IP flow through, “off-beads” and “on-beads” IP elutions. (B) Autoradiograph showing the separation of radiolabeled YBX1 RNP immunoprecipitated from “off-beads” and “on-beads” procedures. Eluted RNPs are separated by SDS-PAGE and transferred to a nitrocellulose membrane. (C-E) Autoradiography of denaturing polyacrylamide gels that are used along the “off-beads” procedure to visualize and select: (C) RNA fragments extracted after Proteinase K digestion of RNPs, (D) products of 3′ adapter ligation, and (E) products of 5′ adapter ligation. (F, G) Agarose gel separation of PCR products at increasing cycle numbers for off-beads (F) and on-beads (G) procedures. For library preparation we chose 15 and 18 cycles, respectively. The expected PCR product runs at ~100 bp. Linker-linker side-products run at 71 bp.
Fig. 3.
Fig. 3.
Overlap of YBX1 binding sites determined by PAR-CLIP using the “off-beads” and the “on-beads” cDNA library preparation procedures. Overlap of at least one nucleotide was determined using the bedtools intersect program using the –u option. Hypergeometric test for the significance of the overlap results in P = 10E-12881.21.
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