Functional coherence of theXistandRSXprotein interactomes: X chromosome inactivation in marsupials

Abstract

AbstractLong 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 (XISTandRSX). TheXistinteractome, comprising proteins that mediate the silencing process, is well documented in mouse studies. Here we interrogate proteins that interact withRSXusing chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) in a marsupial representative,Monodelphisdomestica. We identify 135 proteins that interact withRSX, of which 56 have orthologues in theXistinteractome. Remarkably, nearly 90% of the combinedXistandRSXinteractomes 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 theRSXinteractome were enriched for regions of intrinsic disorder in common with theXistinteractome, identifying this as a feature of ribonucleoprotein complexes associated with XCI. We also show that RNAi knockdown of representativeRSXinteractors, 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 betweenXistandRSX, they exhibit extraordinary functional coherence that indicates potential for post-transcriptional regulation, a feature not previously associated with the molecular machinery of XCI.