PNA interference mapping demonstrates functional domains in the noncoding RNA Xist

Abstract

The noncoding RNA Xist has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of Xist is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of Xist with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.

Figure 1

Description of murine Xist repetitive regions used in this study. Schematic line drawing of Xist. Repetitive sequence regions are colored and labeled with letters A, B, C, and D (3). The majority of Xist transcripts (>90%) do not contain the A region, and thus it was not investigated. Regions C and D are approximately the same size, and antisense oligomers for the C region are directed at the region labeled II and for the D region against the boxed motif.

Figure 2

RNA-FISH of fibroblast cells from various PNA treatments. Female and male fibroblastic cells were treated for 36 h as indicated. After treatment, cells were fixed and processed for RNA-FISH .