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

Abstract

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.

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Figure 1: Discovery of a candidate X-inactivating RNA in the opossum.
Figure 2: Characterization of Rsx RNA.
Figure 3: Links between Rsx RNA expression and X-chromosome inactivation and reactivation.
Figure 4: Autosomal gene silencing in mouse ES cells by an Rsx transgene.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

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

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Acknowledgements

We thank D. Bell and R. Lovell-Badge for advice on the characterisation of Rsx, J. Cloutier and G. Polikiewicz for help with germ-cell preparations and quantitative PCR, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Genomics core (National Institutes of Health, NIH) for RNA sequencing, A. Toth for the HORMAD1 antibody, the Biological and Procedural Services units at the National Institute for Medical Research (NIMR) for animal husbandry and Rsx transgenesis, and J. Cocquet, L. Reynard, H. Byers and members of the Turner and P. Burgoyne laboratories for reading of the manuscript. This work was supported by the Medical Research Council (MRC) (U117588498, U117597141, U117581331, U117597137), the NIH (HD60858), the Robert J. Kleberg Jr and Helen C. Kleberg Foundation, the New Zealand Foundation for Research, Science and Technology, Possum Biocontrol (C10X0501), the Australian National Health and Medical Research Council (1010453) and the NIDDK (NIH) Intramural Research Program.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to James M. A. Turner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4, a Supplementary Discussion and additional references. (PDF 6431 kb)

Supplementary Table

This file contains the probe and primer sequences used in this study. (XLS 46 kb)

Supplementary Table 2

This file contains the female:male ratios for X-encoded transcripts derived from RNA-seq data. (XLS 135 kb)

Supplementary Table 3

This file contains the Australasian marsupial Rsx EST identifiers. (XLS 13 kb)

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Grant, J., Mahadevaiah, S., Khil, P. et al. Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation. Nature 487, 254–258 (2012). https://doi.org/10.1038/nature11171

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