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

Ristová, M., Shchepachev, V., and Tollervey, D.

By Jenna Hare, Bird Lab

Interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. Unfortunately, current methods are not able to capture interactions with naturally unstable proteins, and can recover non-specific interactions. Here, the Tollervey lab present reCRAC (reverse CRAC) as a refined method for mapping protein-RNA interactions in Saccharomyces cerevisiae. It improves upon traditional CRAC (crosslinking and analysis of cDNA) by reversing the steps in the procedure. The main development from the CRAC protocol is the introduction of a strong denaturing step immediately at cell lysis, to minimise such nonspecific interactions and enhance signal quality in a two-step purification. It also makes use of a very small (20 aa) tag, consisting of His8, an Ala4 linker, and a single FLAG epitope. Following the new protocol from the Tollervey lab, samples are lysed directly in cell lysis buffer with 6M Guanidine-HCl and 300mM salt to disrupt weak, non-specific interactions whilst keeping the His-tag accessible. Furthermore, Nickel beads are used which allows for the elution of His-tagged proteins. This is then followed by secondary purification on anti-Flag beads, which pulls down the FLAG-tagged protein of interest.

Researchers from the Tollervey lab validated the effectiveness of this method on the unstable yeast protein, Pin4. Firstly, the RNA binding sites for Pin4 were successfully mapped. Secondly, reCRAC greatly reduced the level of protein degradation compared to standard CRAC procedures. Thirdly, they observed improved cDNA library signal and improved signal-to-noise ratios using the reCRAC method.

This method does require a tagged protein of interest, which may affect expression or RNA-binding efficiency, but this is minimised by the small tag size. reCRAC was shown to reduce degradation and improve detection of interactions of interest. These findings open doors to the study of unstable RNA-interacting proteins which were previously difficult to capture, in yeast and, hopefully, higher organisms.