Dissecting the molecular mechanisms of lncRNA function in X chromosome inactivation across mammalian gestation evolution

Long non-coding RNAs (lncRNAs) are >200-nucleotide transcripts that do not encode proteins, but interact with proteins and regulate gene expression. Whilst full-length sequence conservation is rare for lncRNAs, short regions of higher conservation can exist across species. The XIST lncRNA mediates dosage compensation via X chromosome inactivation (XCI) of a single X chromosome in females. Maintaining gene dosage across sexes is vital for placental mammal prenatal development, failure of which is embryonic lethal. Despite its presence throughout placental mammals, most studies have focused on mouse Xist. It is yet to be determined whether mouse Xist-protein interactions are shared across placental mammals where the timing and nature of XCI differ. Here, we aimed to dissect XIST’s interactors in placental mammals with different implantation strategies. Spen, Hnrnpk, Ciz1, Rbm15 and Wtap proteins were previously identified as mouse Xist functional interactors. Their average amino acid identity is >70% across human, mouse, cow and pig. RT-qPCR and western blotting revealed coordinate expression of XIST and putative protein partners in endometrial tissues/cells from those species. RNA immunoprecipitations showed SPEN, hnRNPK, WTAP and CIZ1 proteins bind human XIST but interactions could not be robustly assessed in cow. RNA pulldowns revealed bovine CIZ1 interacts with XIST E-repeat in cow and RBM15 interacts with human XIST A-repeat, as occurs in mouse. Proteomic analyses indicated hnRNPU and TOP1 bound bovine XIST A-repeat. Bovine XIST A-repeat bound MATR3, RALYL and YY1 in human lysates. Selective pressure variation analyses identified residues under positive selection in a subset of these proteins, but could not explain their differential binding to human and bovine XIST. Altogether, the XIST interactome was characterised for the first time in endometrial-derived bovine cells, revealing cow-specific and conserved interactors across placental mammals. Bovine XIST interactors from human cells reported here may contribute to our understanding of lncRNA-protein partner co-evolution.