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. Researchers from NIAMS 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.
The researchers 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.
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.