reCRAC: A Stringent Method for Precise Mapping of Protein-RNA Interactions in Yeast

Abstract

Intricate interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. UV crosslinking methods to study protein-RNA interactions have been instrumental in elucidating their interactions but can be limited by degradation of target proteins during the process, low signal-to-noise ratios, and non-specific interactions. Addressing these limitations, we describe reCRAC (reverse CRAC), a novel adaptation of the CRAC (crosslinking and analysis of cDNA) technique, optimized for yeast Saccharomyces cerevisiae. Like CRAC, reCRAC applies tandem affinity purification to yield highly enriched protein preparations. However, reCRAC is redesigned to work with unstable proteins. This is achieved by lysing the cells directly into highly denaturing buffer conditions, followed by stringent purification steps. The reCRAC method was successfully applied to the easily degraded yeast protein Pin4, allowing identification of precise binding sites at base-pair resolution with greatly reduced target protein degradation and improved signal-to-noise ratios.