Intersection of the RNA interference and X-inactivation pathways - PubMed (original) (raw)

Intersection of the RNA interference and X-inactivation pathways

Yuya Ogawa et al. Science. 2008.

Abstract

In mammals, dosage compensation is achieved by X-chromosome inactivation (XCI) in the female. The noncoding Xist gene initiates silencing of the X chromosome, whereas its antisense partner Tsix blocks silencing. The complementarity of Xist and Tsix RNAs has long suggested a role for RNA interference (RNAi). Here, we report that murine Xist and Tsix form duplexes in vivo. During XCI, the duplexes are processed to small RNAs (sRNAs), most likely on the active X (Xa) in a Dicer-dependent manner. Deleting Dicer compromises sRNA production and derepresses Xist. Furthermore, without Dicer, Xist RNA cannot accumulate and histone 3 lysine 27 trimethylation is blocked on the inactive X (Xi). The defects are partially rescued by truncating Tsix. Thus, XCI and RNAi intersect, down-regulating Xist on Xa and spreading silencing on Xi.

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Figures

Figure 1

Figure 1. Small RNAs derived from Tsix/Xist

(A) xiRNAs from Xist repeat A (XA) region (map) detected by Northern analysis. Sense (s) and antisense (as) riboprobes detect Tsix and Xist, respectively. miR292-as controls are shown on same blots. M, male. F, female. (B) Northern analysis of xiRNAs from Xist exon 7. (C) Northern analysis of Xist promoter region. (D) Northern analysis of mutant cells. WT lanes identical to those in panel A (concurrent analysis).

Figure 2

Figure 2. Tsix and Xist RNA form long duplexes in vivo

(A) Map of Tsix/Xist and primer pairs (*). (B) In vivo RNAse protection assay. S, sense. AS, antisense. (C) Relative quantities of Xist and Tsix in duplexes measured at position 2 (bp 1206-1337 of Xist) by strand-specific real-time RT-PCR of protected RNA (RNAse+) as compared to total levels (RNAse–). Quantities are standardized to Xist in the Xist-Tsix duplex (Xist, RNAse+ = 1). Error bars=one standard deviation (SD) in triplicate reactions. (D) Quantities of protected Tsix or Xist RNAs (RNase+) relative to total Tsix or Xist (RNase-) for in vivo RNAse protection assays. Error bar=1SD, triplicate reactions. (E) In vivo RNAse protection assays to test allelic origin of dsRNA using strand-specific, allele-specific realtime RT-PCR with SNP-based primers for Xcas or Xmus alleles (Table). PCR of control genomic DNA shows high specificity (98% for mus, >99.99% for cas). Error bar=1SD, triplicate reactions. For test samples, the mus and cas fractions were amplified separately, normalized to genomic DNA (Xcas: Xmus =1:1), and plotted as a function of time. %cas = [Xcas RNA/ (Xcas RNA + Xmus RNA)] × 100. Because the bars show relative allelic fractions, quantities are only comparable within a single timepoint.

Figure 3

Figure 3. Dcr deficiency impairs xiRNA production and XCI

(A) Quantitative realtime RT-PCR of indicated transcripts normalized to β-actin. (B) Xist quantitation plotted on a log scale. (C,D) Northern analyses of miRNA292-as control (C) and xiRNAs (D) in mutant cells. Note accumulation of miRNA292-as precursors (*) in _Dcr_-/- cells. (E) Immuno-RNA FISH for Xist and H3-3meK27 on d10. DAPI, blue. (F) Phase contrast images of d10 EB.

Figure 4

Figure 4. Tsix genetically interacts with Dcr

(A) Allele-specific RT-PCR analysis. All RT- reactions were negative (not shown). Xist d0 samples (asterisks) were 10-fold overloaded to visualize low expression. (B) Realtime RT-PCR of indicated transcripts, each normalized to β-actin. (C) Immuno-RNA FISH for Xist and H3-3meK27 domains (arrowheads) on d10. (D) Frequency of aberrant H3-3meK27 enrichment in the Xist+ subpopulation of indicated cell lines. n=100-150. (E) Model: intersection of RNAi and XCI. (F) Methylation-sensitive restriction analysis of the Xist promoter. Genomic DNA was digested with EcoRV or EcoRV+HpaII. % uncut (methylated) DNA at HpaII is plotted.

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References

    1. Lyon MF. Nature. 1961;190:372. - PubMed
    1. Brown CJ, et al. Cell. 1992;71:527. - PubMed
    1. Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N. Nature. 1996;379:131. - PubMed
    1. Lee JT, Lu N. Cell. 1999;99:47. - PubMed
    1. Fire A, et al. Nature. 1998;391:806. - PubMed

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