X chromosome inactivation in the absence of Dicer

Abstract

Dicer is central to the RNA interference (RNAi) pathway, because it is required for processing of double-stranded RNA (dsRNA) precursors into small RNA effector molecules. In principle, any long dsRNA could serve as a substrate for Dicer. The X inactive specific transcript (Xist) is an untranslated RNA that is required for dosage compensation in mammals. It coats and silences 1 of the 2 X chromosomes in female cells and initiates a chromosomewide change in chromatin structure that includes the recruitment of Polycomb proteins, but it is largely unknown how Xist RNA mediates these processes. To investigate a potential link between the RNAi pathway and X inactivation, we generated and analyzed Dicer-deficient embryonic stem (ES) cells. In the absence of Dicer, coating by Xist RNA, initiation of silencing, and recruitment of Polycomb proteins occur normally. Dicer ablation had modest effects on the steady-state levels of spliced Xist RNA. Together our data indicate that the RNAi machinery is not essential for the initiation of X inactivation.icer is an RNase III-like enzyme involved in the generation of small double-stranded RNAs (dsRNA) from long doublestranded precursors (1, 2). Dicer cleavage products, which are classified as microRNAs (miRNAs) and endogenous small interfering RNAs (esiRNAs), are then bound by Argonautes (Ago) within multiprotein complexes and can regulate gene expression via several distinct mechanisms (3, 4). miRNAs have been extensively characterized (5), and the majority of miRNAs mediate posttranscriptional gene silencing (PTGS) by inhibiting translation of cognate mRNAs and/or promoting mRNA decay (6, 7). esiRNAS can be generated by transcription of sense and antisense transcripts, which can then form dsRNA and get processed by Dicer. The characterization of esiRNAs is currently less advanced (8). They have been cloned from the germ line of mammalian organisms (9-11), and their existence in somatic cells is anticipated (12) because any long double-stranded RNA in a cell could, in principle, be processed by Dicer.One process that involves long, untranslated RNAs is the silencing of the X chromosome in female mammalian cells. The X inactive specific transcript (Xist), is a nuclear RNA transcribed from the inactive X chromosome (Xi). In female mammals, Xist RNA is absolutely required for dosage compensation, which is initiated during early embryogenesis (13-15). Upon counting of the X chromosomes, 1 X is designated to remain active (Xa), and Xist is transcriptionally up-regulated on the other X, which is destined to be inactivated. The Xist RNA accumulates on this chromosome in cis and mediates transcriptional gene silencing through unknown mechanisms. Polycomb group (PcG) proteins are recruited to the Xist RNA-associated chromosome and establish chromosomewide histone modifications that include histone H3 lysine 27 trimethylation (16,17). Later in the process, additional chromatin marks, ranging from H3K9 and H4K20 methylation to DNA methylation and histone variant recruitment, become enrich...

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“…Oligo sequences used are described in Table S1. ES cell derivation was performed according to standard protocols (52). Pregnant mice from timed matings were killed at indicated time points by CO 2 asphyxiation following institutional guidelines.…”

X chromosome inactivation sparked by non-coding RNAs

“…11 These results did not remain unchallenged and follow-up studies have shown that X inactivation can occur in Dicer deficient embryos indicating that the RNAi pathway plays no critical role in X inactivation. 51 Xist upregulation was observed in a low percentage of Dicer deficient cells. This can most likely be explained by an indirect effect caused by the downregulation of Dnmts as a consequence of Dicer deficiency.…”

The lesser known story of X chromosome reactivation

“…Dicer could be involved in downregulating Xist in the Xa, 63 but another report showed that, while Xist is not detected in Dicerknockout female mESCs, this is most likely due to aneuploidy caused by X instability rather than an effect of Dicer. 64 The claim is substantiated by the lack of small RNA remnants of Xist/Tsix duplex and the normal XCI found in hybrid ES cells. More studies on the role of RNAi processing triggered by the complementarity of Xist/Tsix are needed, particularly because this pathway is a logically relevant player in XCI.…”