Capturing the Interactome of Newly Transcribed RNA (PAR-CLIP)

Key features of gene transcription, such as transcriptional pausing, divergent transcription, and enhancer RNA production can be explored using techniques based on the incorporation of synthetic nucleoside analogs into RNA, followed by pull-down and high-throughput sequencing. Whether these approaches can be adapted to expand our knowledge of the RNA-bound proteome is, however, unexplored. Here, we have designed a methodology (isolation of the newly transcribed RNA Interactome using ClicK reaction; RICK) based on the incorporation of 5-ethynyluridine (EU) into newly transcribed RNAs to systematically capture proteins bound to RNAs of wide scope including traditionally neglected non-polyadenylated RNAs. Amongst the novel RNA-binding proteins identified by RICK, we found mitotic regulators, components of the DNA damage response pathway, and epigenetic regulators. Because the protocol can be easily modified to include variations such as labeling time or transcriptional interference, RICK will enable an in depth and global interrogation of the RNA-bound proteome. Perform 2 independent PAR-CLIP sequencing experiments for CDK1 and METTL1 in HEK293T cells