Identification of proteins directly interacting with the HELLP-syndrome associated lincRNA using ChIRP

The HELLP syndrome is a pregnancy-associated disease inducing hemolysis, elevated liver enzymes, and low platelets in the mother. Although the HELLP symptoms occur in the third trimester in the mother, the origin of the disease can be found in the first trimester fetal placenta. The chromosomal locus on 12q23.2 between PMCH and IGF1 with linkage to the HELLP syndrome contains a 205 kbp long intergenic noncoding RNA with (peri)nuclear expression in the extravillous trophoblast. siRNA-mediated knockdown followed by RNA-sequencing revealed that the HELLP lincRNA activated a large set of genes that are involved in the cell cycle. Furthermore, blocking potential mutation sites identified in HELLP families decreased the invasion capacity of extravillous trophoblasts. Because lincRNAs predominantly function through interaction with proteins, we used a method named Chromatin Isolation by RNA Purification (ChIRP) to identify proteins directly interacting with the HELLP lincRNA. The method was optimized using the KCNQ1OT1 noncoding RNA interacting with DNMT1. Cultured SGHPL-5 extravillous trophoblast cells were crosslinked, and the lysed complexes DNAse treated. To capture the HELLP lincRNA, four biotinylated morpholino probes were used. The HELLP and negative controle morpholinos were hybridized and isolated using magnetic straptavidin beads. Extracted proteins were analysed by protein mass spectrometry. 22 proteins were only present in the ChIRP sample treated with HELLP lincRNA morp...
Source: Placenta - Category: Reproduction Medicine Authors: Tags: Frontmatter Source Type: research