Abstract

Long range epigenetic silencing is epitomised by X chromosome inactivation (XCI) in mammals. It is mediated by independently evolved, non-homologous long noncoding RNAs in eutherian and marsupial mammals (XIST and RSX). The Xist interactome, comprising proteins that mediate the silencing process, is well documented in mouse studies. Here we interrogate proteins that interact with RSX using chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) in a marsupial representative, Monodelphis domestica. We identify 135 proteins that interact with RSX, of which 56 have orthologues in the Xist interactome. Remarkably, nearly 90% of the combined Xist and RSX interactomes are within the same protein-protein association network. This network clustered into three major groups with distinctive functional enrichments, including RNA splicing and processing, regulation of translation and ribosomal biogenesis, and epigenetic transcriptional silencing. The proteins of the RSX interactome were enriched for regions of intrinsic disorder in common with the Xist interactome, identifying this as a feature of ribonucleoprotein complexes associated with XCI. We also show that RNAi knockdown of representative RSX interactors, HNRNPK and CKAP4, led to reactivation of transcription from the inactive X chromosome, indicating a role for each in marsupial XCI. Thus, despite the absence of linear sequence homology between Xist and RSX, they exhibit extraordinary functional coherence that indicates potential for post-transcriptional regulation, a feature not previously associated with the molecular machinery of XCI.