Bag Indira, Chen Yang, D'Orazio Karole, Lopez Prisma, Wenzel Sabine, Takagi Yuichiro, Lei Elissa P
Nuclear Organization and Gene Expression Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
G3 (Bethesda). 2022 Aug 25;12(9). doi: 10.1093/g3journal/jkac152.
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.
染色质绝缘子是遍布基因组的DNA-蛋白质复合物，能够建立独立的转录结构域。此前有报道称，果蝇的su(Hw) mRNA在细胞核内与吉普赛染色质绝缘子蛋白复合物发生物理结合，可能发挥非编码功能来影响绝缘子活性。然而，这种mRNA是如何被招募到吉普赛复合物中的尚不清楚。在这里，我们利用RNA亲和拉下结合质谱法鉴定了一种新的RNA结合蛋白Isha（CG4266），它在体外和体内均与su(Hw) mRNA结合。Isha具有一个保守的RNA识别基序和RNA聚合酶II C末端结构域相互作用结构域（CID）。我们发现Isha与全酶和延伸中的聚合酶II发生物理相互作用，并以RNA依赖的方式与基因5'端的染色质结合。此外，ChIP-seq分析显示Isha在染色质上特别与核心吉普赛绝缘子成分CP190重叠。Isha的缺失会降低吉普赛绝缘子的增强子阻断和屏障活性，并破坏绝缘子体的核定位。我们的结果揭示了一种新的因子Isha，它促进吉普赛绝缘子活性，可能作为su(Hw)非编码mRNA的核RNA结合蛋白适配器。
