Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation

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In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY)1. X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist2. Xist is not found in metatherians3 (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades4. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.

At a glance


  1. Discovery of a candidate X-inactivating RNA in the opossum.
    Figure 1: Discovery of a candidate X-inactivating RNA in the opossum.

    a, RNA FISH mapping of the new gene using BACs; green BACs give RNA FISH cloud signals, red BACs do not. The VM18-839J22 BAC gives a cloud signal (green; second panel, bottom) identical to Xist RNA (first panel, bottom) as seen in mouse brain cells (further RNA cloud images in Supplementary Fig. 1). The cloud is still observed with VM18-839J22del (third panel, bottom), deleted for Hprt1 (Supplementary Table 1 for recombineering sequences) and VM18-303M7 (fourth panel, bottom). The 82-kb critical interval is defined by VM18-839J22del and VM18-3O1. The Lnx3 gene that gave rise to Xist3 maps to a locus distinct from Rsx. DAPI, 4′,6-diamidino-2-phenylindole; Mb,megabases. b, Top, RT–PCR using primers denoted ‘L’ (originating in the left half of the critical region in the figure) and ‘R’ (originating in the right half of the critical region in the figure) identifies a female-specific 47-kb transcript (green boxed area in RT–PCR figure) within the 82-kb critical interval (see Supplementary Table 1 for primers). Transcript limits are shown above the X chromosome. Middle, RNA FISH images showing RNA clouds in female but not male brain cells. Bottom, RT–PCR using primer pair M3 (black rectangle in first RT–PCR image) shows female-specific expression in all tissues. Gapdh is an autosomal control. c, Combined VM18-839J22 RNA FISH and H3K27me3 immunostaining shows the inactive X chromosome (marked by dotted line in first panel) coated with the new RNA. d, Combined VM18-839J22 RNA and DNA FISH for the new RNA. No RNA signal is observed from the active X chromosome (Xa) locus, but an RNA signal colocalizes with the DNA locus on the inactive X chromosome (Xi). Scale bars, 5μm.

  2. Characterization of Rsx RNA.
    Figure 2: Characterization of Rsx RNA.

    a, RNA-seq shows female-specific expression of Rsx, whereas reads mapping to the Phf6 and Hprt1 genes are found in both sexes. Boxed area shows magnification of Rsx locus. Pale red reads are ambiguous, that is, they hit several repeats within the Rsx RNA. Inferred exons and their verification by RT–PCR are shown at the bottom. Dark grey bars along the chromosome coordinate axis represent DNA sequence gaps (Methods). b, Northern blot analysis of Rsx. (See Supplementary Fig. 2 for controls, and Supplementary Table 1 for primers for probes.) Top left, female-specific expression of an RNA greater than 23kb. Middle, size verification of Rsx RNA by comparison with the 17-kb Xist RNA. Verification of the strandedness of Rsx transcription (sequences in Supplementary Table 1) is shown in the right two panels. Bottom, multi-tissue blot showing female-specific Rsx expression in all tissues. c, Comparison of repeat organization with that of Xist by sequence-similarity plots, window size = 28 nucleotides (grey area represents the unsequenced 2.8kb). The 5′ 12-kb stretch of repeats includes two highly conserved 34- and 35-base motifs (Supplementary Fig. 3 for predicted 34-base stem–loops). RNA FISH using a repeat probe (green) co-localizes with the VM18-839J22 BAC (red; antisense probe sequence, Supplementary Table 1). A sense probe generates no signals (data not shown), confirming the transcriptional orientation of Rsx. Scale bar, 5μm. d, Adjusted female:male ratios inferred from brain RNA-seq data identifying Rsx as a candidate XCI RNA (Methods). The second highest ranking RNA, ENSMODG00000003195, was found by RT–PCR to not be female-specific in other tissues, so can be excluded as an XCI candidate.

  3. Links between Rsx RNA expression and X-chromosome inactivation and reactivation.
    Figure 3: Links between Rsx RNA expression and X-chromosome inactivation and reactivation.

    a, Rsx clouds (arrowheads) are present in supporting cells (s) but not in meiotic cells (m, labelled with HORMAD1). b, Rsx clouds (in lower panel; arrowheads) colocalize with the XCI marker H3K27me3, which is not observed in meiotic cells. c, RNA FISH for the X-chromosome gene Msn shows that although supporting cells undergo XCI (that is, display a single RNA spot, arrows), meiotic cells have two active X chromosomes. Msn RNA signals are very bright in meiotic cells owing to an increase in global transcription during this point in germ-cell development. Scale bars, 5μm.

  4. Autosomal gene silencing in mouse ES cells by an Rsx transgene.
    Figure 4: Autosomal gene silencing in mouse ES cells by an Rsx transgene.

    a, In Rsx transgenic female ES cell clone 303.2, the full-length Rsx transgene is expressed, as shown by RT–PCR spanning all exon–exon boundaries and by M3 (see Fig. 1) RT–PCR. b, Rsx RNA appears as a cloud in 303.2 cells, indicating autosomal coating. RNA FISH for three chromosome 18 genes, Ndfip1, Prrc1 and Synpo, shows that this coating induces gene silencing in differentiated cells (left column), whereas in other differentiated cells silencing does not occur (middle column). Wild-type (WT) ES cells show biallelic expression for each chromosome 18 gene (right column). TG, transgenic. Scale bars, 5μm. c, Quantification of gene silencing (n>100 cells per gene) in differentiated 303.2 cells versus controls (see Supplementary Table 1 for PCR primers for chromosome 18 RNA FISH probes). RNA FISH for the chromosome 9 gene Atr serves as a positive control.

Accession codes

Primary accessions

Gene Expression Omnibus


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Author information


  1. MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

    • Jennifer Grant,
    • Shantha K. Mahadevaiah,
    • Mahesh N. Sangrithi,
    • Hélène Royo,
    • Willie Taylor,
    • Greg Elgar,
    • Mike J. Gilchrist &
    • James M. A. Turner
  2. National Institute of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892, USA

    • Pavel Khil &
    • R. Daniel Camerini-Otero
  3. Landcare Research - Manaaki Whenua, Pest Control Technology Group, Lincoln 7640, New Zealand

    • Janine Duckworth
  4. University of Texas at San Antonio, San Antonio, Texas 78249, USA

    • John R. McCarrey
  5. Texas Biomedical Research Institute, San Antonio, Texas 78227, USA

    • John L. VandeBerg
  6. Department of Zoology, University of Melbourne, Victoria, Australia 3010

    • Marilyn B. Renfree


J.G. and J.M.A.T. conceived and designed the experiments, performed RNA FISH and RT–PCR and wrote the manuscript. P.K., R.D.C.-O. and M.J.G. generated and analysed RNA-seq data. J.G., G.E. and W.T. performed repeat analysis. J.M.A.T. and S.K.M. performed northern blots. M.N.S. generated the transgenic ES cell line, and H.R. determined the ES cell transgene copy number. J.D., J.R.M., J.L.V. and M.B.R. provided animals and tissues.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

RNA-seq data is available from the Gene Expression Omnibus under accession number GSE36861; Rsx accession number JQ937282.

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Supplementary information

PDF files

  1. Supplementary Information (6.2M)

    This file contains Supplementary Figures 1-4, a Supplementary Discussion and additional references.

Excel files

  1. Supplementary Table (46K)

    This file contains the probe and primer sequences used in this study.

  2. Supplementary Table 2 (135K)

    This file contains the female:male ratios for X-encoded transcripts derived from RNA-seq data.

  3. Supplementary Table 3 (13K)

    This file contains the Australasian marsupial Rsx EST identifiers.

Additional data