RNA helicase Ddx5 and the noncoding RNA SRA act as coactivators in the Notch signaling pathway

Notch signaling plays a pivotal role in embryonic and postnatal development. Upon binding of a Notch ligand, proteolytic cleavage events liberate the Notch-intracellular domain (NICD) that migrates into the nucleus. In order to activate target genes, NICD associates with the transcription factor RBP-J (also known as CSL), Mastermind and the acetyltransferase p300. Here, we identify the DEAD-box RNA helicase Ddx5 as a novel component of the RBP-J/NICD complex utilizing a biotinylation-tagging approach followed by mass-spectrometry. Biochemical assays confirm a direct interaction of Ddx5 with RBP-J. We show that Ddx5 localizes at RBP-J binding sites within the Notch target genes preTCRa, Hesl and CD25 in a Notch-dependent manner. Moreover, knockdown of Ddx5 also downregulates a subset of Notch target genes in a murine pre 1-cell model. Interestingly, also knockdown/overexpression of the RNA coactivator SRA, a cofactor of Ddx5, downregulates Hesl and preTCR alpha. Using Chromatin-IP, we show that this effect is accompanied with a loss of p300 occupancy at Notch target genes and decreased histone acetylation. Together, our data demonstrate that Ddx5 and SRA function as coactivators of Notch signaling. (C) 2013 Elsevier B.V. All rights reserved.