Abstract
Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.