Genome-wide Identification of DEAD-box RNA Helicase Targets Reveals Roles for RNA Secondary Structure Remodeling in mRNA Processing (iCLIP-seq)

Accurate gene expression requires the coordination of RNA processing with assembly of messenger RNA-protein (mRNP) complex. RNA helicases are a class of enzymes that unwind RNA duplexes in vitro and have been are proposed to remodel RNA structure in vivo. Herein, we provide evidence that the DEAD-box protein Dbp2 remodels RNA structure to facilitate efficient pre-mRNA processing in S. cerevisiae. First, we find that Dbp2 associates with the 3' ends and 3' splice-sites of mRNAs genome-wide. Using structure-seq to map RNA secondary structure, we find altered secondary structures in dbp2? cells that overlap polyadenylation elements and correlate with inefficient termination. We also identify a role for Dbp2 in pre-mRNA splicing and show that both splicing and termination require Dbp2 helicase activity. This reveals that DEAD-box RNA helicases unwind structure in vivo and that structural alteration of pre-mRNA is essential for proper gene expression. Overall design: Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) of Dbp2 and Set1 in Saccharomyces cerevisiae. 3 biological replicates for both Dbp2- and Set1-iCLIP. Set1-iCLIP serves as a negative control in this study.