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Targeting Xist with compounds that disrupt RNA structure and X inactivation


Although more than 98% of the human genome is non-coding1, nearly all of the drugs on the market target one of about 700 disease-related proteins. The historical reluctance to invest in non-coding RNA stems partly from requirements for drug targets to adopt a single stable conformation2. Most RNAs can adopt several conformations of similar stabilities. RNA structures also remain challenging to determine3. Nonetheless, an increasing number of diseases are now being attributed to non-coding RNA4 and the ability to target them would vastly expand the chemical space for drug development. Here we devise a screening strategy and identify small molecules that bind the non-coding RNA prototype Xist5. The X1 compound has drug-like properties and binds specifically the RepA motif6 of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that RepA can adopt multiple conformations but favours one structure in solution. X1 binding reduces the conformational space of RepA, displaces cognate interacting protein factors (PRC2 and SPEN), suppresses histone H3K27 trimethylation, and blocks initiation of X-chromosome inactivation. X1 inhibits cell differentiation and growth in a female-specific manner. Thus, RNA can be systematically targeted by drug-like compounds that disrupt RNA structure and epigenetic function.

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Fig. 1: X1 binds RepA and weakens RNA binding to interacting protein partners in vitro.
Fig. 2: X1 weakens the Xist interaction with PRC2 and SPEN in a RepA-dependent manner inside cells.
Fig. 3: X1 treatment leads to Xi-specific loss of PRC2 and H3K27me3 enrichment and failure of XCI.
Fig. 4: 3D structure of RepA with and without X1.

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Data availability

Sequence data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) with accession number GSE141683.


  1. Consortium, E. P. et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).

    Article  ADS  Google Scholar 

  2. Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017).

    Article  CAS  Google Scholar 

  3. Warner, K. D. et al. Principles for targeting RNA with drug-like small molecules. Nat. Rev. Drug Discov. 17, 547–558 (2018).

    Article  CAS  Google Scholar 

  4. Zhang, F. & Lupski, J. R. Non-coding genetic variants in human disease. Hum. Mol. Genet. 24, R102–R110 (2015).

    Article  CAS  Google Scholar 

  5. Lee, J. T. Epigenetic regulation by long noncoding RNAs. Science 338, 1435–1439 (2012).

    Article  ADS  CAS  Google Scholar 

  6. Wutz, A. et al. Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat. Genet. 30, 167–174 (2002).

    Article  CAS  Google Scholar 

  7. Rizvi, N. F. & Smith, G. F. RNA as a small molecule druggable target. Bioorg. Med. Chem. Lett. 27, 5083–5088 (2017).

    Article  CAS  Google Scholar 

  8. Disney, M. D. et al. Drugging the RNA world. Cold Spring Harb. Perspect. Biol. 10, a034769 (2018).

    Article  CAS  Google Scholar 

  9. Howe, J. A. et al. Selective small-molecule inhibition of an RNA structural element. Nature 526, 672–677 (2015).

    Article  ADS  CAS  Google Scholar 

  10. Palacino, J. et al. SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nat. Chem. Biol. 11, 511–517 (2015).

    Article  CAS  Google Scholar 

  11. Disney, M. D. et al. Inforna 2.0: a platform for the sequence-based design of small molecules targeting structured RNAs. ACS Chem. Biol. 11, 1720–1728 (2016).

    Article  CAS  Google Scholar 

  12. Allen Annis, D. et al. An affinity selection-mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries—Discovery of a novel antagonist of E-coli dihydrofolate reductase. Int. J. Mass Spectrom. 238, 77–83 (2004).

    Google Scholar 

  13. Rizvi, N. F. et al. Discovery of selective RNA-binding small molecules by affinity-selection mass spectrometry. ACS Chem. Biol. 13, 820–831 (2018).

    Article  CAS  Google Scholar 

  14. Rizvi, N. F. et al. Targeting RNA with small molecules: identification of selective, RNA-binding small molecules occupying drug-like chemical space. SLAS Discov. 25, 384–396 (2020).

  15. Cifuentes-Rojas, C. et al. Regulatory interactions between RNA and polycomb repressive complex 2. Mol. Cell 55, 171–185 (2014).

    Article  CAS  Google Scholar 

  16. Chillon, I. et al. Native purification and analysis of long RNAs. Methods Enzymol. 558, 3–37 (2015).

    Article  CAS  Google Scholar 

  17. Lipinski, C. A. Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov. Today Technol. 1, 337–341 (2004).

    Article  CAS  Google Scholar 

  18. Monfort, A. et al. Identification of Spen as a crucial factor for Xist function through forward genetic screening in haploid embryonic stem cells. Cell Rep. 12, 554–561 (2015).

    Article  CAS  Google Scholar 

  19. Lee, M. K. et al. A novel small-molecule binds to the influenza A virus RNA promoter and inhibits viral replication. Chem. Commun. 50, 368–370 (2014).

    Article  CAS  Google Scholar 

  20. Ogawa, Y. et al. Intersection of the RNA interference and X-inactivation pathways. Science 320, 1336–1341 (2008).

    Article  ADS  CAS  Google Scholar 

  21. Zhao, J. et al. Genome-wide identification of polycomb-associated RNAs by RIP–seq. Mol. Cell 40, 939–953 (2010).

    Article  CAS  Google Scholar 

  22. Patil, D. P. et al. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537, 369–373 (2016).

    Article  ADS  CAS  Google Scholar 

  23. Sunwoo, H. et al. Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1). Proc. Natl Acad. Sci. USA 114, 10654–10659 (2017).

    Article  CAS  Google Scholar 

  24. Jeon, Y. & Lee, J. T. YY1 tethers Xist RNA to the inactive X nucleation center. Cell 146, 119–133 (2011).

    Article  CAS  Google Scholar 

  25. Colognori, D. et al. Xist deletional analysis reveals an interdependency between Xist RNA and polycomb complexes for spreading along the inactive X. Mol. Cell 74, 101–117 e110 (2019).

    Article  CAS  Google Scholar 

  26. Liu, F. et al. Visualizing the secondary and tertiary architectural domains of lncRNA RepA. Nat. Chem. Biol. 13, 282–289 (2017).

    Article  Google Scholar 

  27. Kikhney, A. G. & Svergun, D. I. A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins. FEBS Lett. 589, 2570–2577 (2015).

    Article  CAS  Google Scholar 

  28. Kim, D. N. et al. Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution. Nat. Commun. 11, 148 (2020).

    Article  ADS  CAS  Google Scholar 

  29. Carrette, L. L. G. et al. A mixed modality approach towards Xi reactivation for Rett syndrome and other X-linked disorders. Proc. Natl Acad. Sci. USA 115, 1715124115 (2017).

    Google Scholar 

  30. Stelzer, A. C. et al. Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble. Nat. Chem. Biol. 7, 553–559 (2011).

    Article  CAS  Google Scholar 

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We thank all members of the Lee and Patel laboratories, Charles Lesberg and other Merck team members for stimulating scientific discussions. We acknowledge the Merck MINt Award, a grant from the US National Institutes of Health (R01-HD097665), and funding from the Howard Hughes Medical Institute to J.T.L.; the Pew Charitable Trust Latin American Fellows Program to R.A. and C.R.; and the MGH Fund for Medical Discovery to R.A. M.D.B. and T.M. acknowledge MITACS and NSERC PGS-D fellowships respectively. T.R.P. is Canada Research Chair in RNA and Protein Biophysics. We thank DIAMOND Light Source B21 beamline staff for their help with data collection (BAG SM22113).

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Authors and Affiliations



R.A., K.S., B.K., N.R., T.M., M.D.B., J.M., G.S., J.B., P.J.D., T.R.P., E.N. and J.T.L. designed experiments and interpreted data. R.A. performed RNA purifications, biochemical assays, immunoFISH, ChIP–seq and RNA-seq. K.S. coordinated the small molecule synthesis and off-target activity evaluations. N.R. and E.N. performed the ALIS determinations. C.R. performed RNA purifications. B.K. conducted bioinformatics analyses. T.M., M.D.B. and T.R.P. performed and analysed SAXS data. R.A., K.S. and J.T.L. wrote the paper.

Corresponding author

Correspondence to Jeannie T. Lee.

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Competing interests

K.B.P., N.F.R., J.D.M., G.F.S., J.B., P.J.D. and E.B.N. are current or former employees of Merck & Co. and may hold stock or other financial interests in Merck & Co. J.T.L. is a cofounder of Translate Bio and Fulcrum Therapeutics and is also a scientific advisor to Skyhawk Therapeutics.

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Extended data figures and tables

Extended Data Fig. 1 Purification of Xist RepA RNA.

A 431 Repeat A fragment of Xist RNA was in vitro transcribed and purified under native conditions by FPLC. A representative chromatogram is shown. To confirm size and stability of the sample just prior to ALIS, we visualized the RNA in a denaturing urea-PAGE.

Extended Data Fig. 2 X1 inhibits interaction of Xist RepA with cognate interacting proteins in vitro.

a, RNA EMSAs show that X1 weakens interaction between RepA and PRC2, and RepA and SPEN-RRM. Increasing concentrations of the compounds (0, 5, 7.5, 10, 25, 50, 75, 100 μM) were titrated against 0.5 nM RNA and 15.6 nM PRC2, or 0.1 nM RNA and 158 nM SPEN-RRM. Two replicates showed similar results. b, RNA EMSAs titrating PRC2 (0, 15.6, 31.2, 62.4, 124.9, 250 nM) or SPEN-RRM (0, 79.2, 158, 396, 792, 1580 nM) against a fixed concentration of X1 (25 or 75 μM) and 0.5 nM RepA, Tsix (reverse complement of RepA), or Hotair RNA—all of which are known PRC2 interactors. For SPEN, RNA was 0.1 nM. Two or more replicates showed similar results. c, Increasing concentrations of X1 (0, 5, 7.5, 10, 25, 50, 75, 100 μM) was titrated against 0.5 nM RNA (Tsix, Hotair) and 15.6 nM PRC2. One representative gel of two replicates is shown. d, Densitometric analysis to determine IC50, which were too high to be measured for Tsix and Hotair. Data are represented as mean +/− SD. n = 2 independent experiments. RepA result from Fig. 1f is shown as reference. e, Order of addition does not affect X activity. Increasing concentrations of the compounds (0, 5, 7.5, 10, 25, 50, 75, 100 μM) was titrated against 0.5 nM RepA and 15.6 nM PRC2. One representative gel of two replicates is shown. Top, PRC2 was added to a RepA-molecule pre-incubated mix. Bottom, Molecule was added to a RepA-PRC2 pre-incubated mix.

Extended Data Fig. 3 X1 also inhibits interaction of Xist RepA with cognate interacting proteins in vivo.

a-b, RIP-qPCR analysis in d4 female TST ES cells to evaluate Xist binding to EZH2 (a) and RBM15 (b) in 10 μM X1. IgG, negative control antibody. Other EZH2 interactors Malat1, Gtl2, Htr6-us and Nespas are shown. Gapdh, negative control RNA. Bars: mean. Individual data points included. n = 2 biologically independent experiments quantified in duplicate (Representative graph shown). c, Top: RT-qPCR confirms similar quantities of Xist RNA in control and X-A samples prior to Xist RNA pulldown. Xist exons 4-5 primers were used. Bottom: Similar quantities were also present following Xist RNA pulldown, thereby ruling out unequal Xist expression as a cause of unequal H3 radioactive counts. X-A cells amplified poorly with RepA primers, consistent with deletion of RepA.

Extended Data Fig. 4 X1 effects on EB outgrowth in ♀- TST-XX, ♀-XO, and ♂-XY EB cells.

a, Growth of differentiating ♀-TST cells at day 3, or 24 h post-X1 treatment, up to 10 μM X1. Data are represented as Tukey box plots. Lower whisker: 25th percentile minus 1.5xInterquartile Range (IQR). Higher whisker: 75th percentile plus 1.5xIQR. Box range: 25th (bottom) to 75th (top) percentile. Line within box: median. Points beyond higher whisker are shown. P-values: one-way ANOVA with respect to control cells. n = 150 colonies combined from 3 independent experiments. b, Viability of d5 cells. n = 3 biologically independent experiments. c, No obvious effect on day 3 female EB growth after 24 h X1 treatment. d, Quantitation of EB outgrowth at day 5 (72 h post-drug application). The distance from EB center to edge of outgrowth was measured in 100 d3 or 30 d5 EBs combined from 3 independent experiments. Data presented as in panel (a). P-values: one-way ANOVA with respect to control cells. e, Weaker effect of X16 on ♀-TST EB outgrowth. No obvious effect of X-negative. One representative brightfield microscopy from 3 independent cultures is shown. Center of the EB and edge of outgrowth as marked. Scale as indicated. f, X1 had no effect on growth of pre-XCI (d0) female cells. g,h, X1 also did not inhibit ♀-TST-XO and ♂-XY ES cells at day 3 (g) or day 5 (h). Neither cell line expresses Xist or undergoes XCI. One representative field is shown. Scale bar, 150 μm. i, Quantitation of EB outgrowth in XY male and XO female EBs at days 3 and 5. Distance from the EB center to the edge of outgrowth was measured. Day 3: n = 136, XO colonies; n = 112, XY colonies. Day 5: n = 40, XO colonies; n = 60, XY colonies). Data presented as in panel (a).

Extended Data Fig. 5 Karyotype analysis of ES cells and RNA immunoFISH analysis of day 3 X1-treated cells.

a, X-chromosome painting DNA FISH of DMSO- and X1-treated XX TST cells, and a DMSO-treated XO clone that spontaneously arose from the XX TST cells. Scale as shown. Inset: magnification of representative nucleus. %nuclei with indicated X chromosome number shown. n, sample size combining from 3 biologically independent experiments. b, Xist/Tsix RNA-FISH and immunostaing for H3K27me3, H2AK119ub, EZH2, and RING1B in ♀-TST EB at day 3. One representative nucleus is shown. %cells with Xist foci is indicated. n, sample size. Scale bar, 5 μm.

Extended Data Fig. 6 Full fields for RNA immunoFISH experiments of Fig. 2 and Extended Data Fig. 5.

a, Full fields for the RNA FISH and Immunofluorescence experiments, with boxed nuclei presented in Fig. 2g and Extended Data Fig. 5b. b, Full fields for H3K27me3 immunostaining of DMSO- or X1-treated ♀ TST-A cells, with boxed nuclei presented in Fig. 2h. %cells with foci on the Xi as indicated (sample size, n, from two biologically independent experiments combined). c, Western blot using H3K27me3 and total histone H3 antibodies. Total cell extracts were obtained from day 7 female EB cells after treating with 10 μM of various compounds from day 2. Compounds: EZH2 inhibitor 1 (EPZ-6438, MedChem Express), EZH2 inhibitor 2 (PF-06821497, Pfizer), or X1. One representative film of two replicates is shown.

Extended Data Fig. 7 Epigenomic analyses of PRC2 and H3K27me3 enrichment.

a–b, Allele-specific H3K27me3 (a) and SUZ12 (b) ChIP-seq analyses of day 5 female EB treated with 10 μM X1 or DMSO (control) for 72 h. Tracks for all reads (composite, "comp"), mus (Xi), and cas (Xa). Dotted green lines separate ChrX. c–f, Zoom-ins for allele-specific H3K27me3 ChIP-seq analyses of day 5 female EB treated with 10 μM X1 or DMSO (control) for 72 h. Browser shots shown with sliding window 1 kb, step size 0.5 kb. Scale shown in brackets. c, X-linked genes subjected to XCI. d, the Xist gene. e, Escapees. f, Representative control autosomal gene on Chr13. g, Box plot of normalized read densities for the −5000 to +1 region of ChrX and Chr13 refSeq genes, parsed into mus and cas alleles. Lower whisker: 10th percentile. Higher whisker: 90th percentile. Box range: 25th (bottom) to 75th (top) percentile. Line within box: median. Points beyond whiskers are shown. P-values: two-tailed Wilcoxon test from data gathered from individual H3K27me3 and Suz12 ChIP-seq experiments.

Extended Data Fig. 8 Analysis of gene expression and X1 reversibility.

a, Time course RT-qPCR of indicated control genes in DMSO- or X1-treated female EB. X1 added on indicated days (pink arrows). Mean and S.D. shown for 3 biological replicates. b, Time course allele-specific RT-qPCR of indicated Xa genes in DMSO- or X1-treated female EB. X1 added on indicated days (pink arrows). Mean and S.D. shown for 3 biological replicates. c, Dose-response analysis in the range of 0–10 μM X1 compound. Allele-specific RT-qPCR of indicated X-linked genes in DMSO- or X1-treated female EB. Mus allele (Xi) shown. X1 was added on d2. P, two-tailed Student’s t-test with respect to DMSO-treated TST control. Mean and S.D. shown for 2 replicates. At 10 μM X1, the Student’s t-test reveal no significant difference between d7 cells and expression found in control ES cells. d–e, Female EB were grown from d1 in 10 μM X1 and the treatment was suspended on day 3, 4, 5, or maintained up to day 7. The growth morphology (d) and Mecp2 expression from the Xi is evaluated at d7 (e). One representative brightfield microscopy from 3 independent cultures is shown.

Extended Data Fig. 9 Transcriptomic studies of on- and off-target effects.

a–d, RNA-seq analyses of day 5 DMSO- or X1-treated female EB. Zoom-ins to representative X-linked genes subjected to XCI (a), Xist (b), escapee gene (c) and autosomal gene (d). Tracks for all reads (comp), mus reads (Xi), and cas reads (Xa). FPM scale shown in brackets. e, Differentially expressed autosomal genes (y axis) and their corresponding changes in H3K27me3 enrichment (x axis). Each dot represents a gene. Number of genes on each of the nine sections as shown. Comp tracks were sampled to the smallest library, then MultiTesting and IndependentFiltering DESeq2 filtering was performed reporting significance below 0.05 (Wald test) after Benjamini and Hochberg correction with the application of independent intensity filtering.

Extended Data Fig. 10 Sixteen conformational clusters identified for native RepA RNA without X1 treatment.

a, HPLC-SEC profile of the purified RepA RNA with or without X1 previous to SAXS data collection. b, PRIMUS analysis for initial data quality analysis. Inset: Guinier plot to determine the Radius of Gyration (Rg). c, Dimensionless Kratky analysis [q*Rg vs. I(q)/I(0)x(q*Rg)2] of samples. d, Pairwise distance distribution profile (P(r)) to estimate the real space dimensions of the molecule in Å. e, 16 clusters (C1–C16) of RepA are presented in their native state without X1. C13 is the dominant conformation. Pie-chart shows relative abundance of structural clusters. See also Fig. 4.

Supplementary information

Supplementary Information


Reporting Summary

Supplementary Figure 1

Original source images for EMSAs and western blots.

Supplementary Table 1

RNAs tested in ALIS surveys. The 42 transcripts tested against X22 or X1 are listed. The mouse Xist RepA RNA (the only X22 binder) is highlighted in yellow. RNAs with >60% GC content are highlighted in pink.

Supplementary Table 2

Analogue compounds from Fig. 1c that demonstrated binding in affinity ranking experiments.

Supplementary Table 3

Cellular uptake and concentration of X1 molecule by up to 180-fold. Here we addressed whether cells concentrated the X1 molecule following uptake. In brief, we added a known amount of tritiated X1 molecule to embryonic stem (ES) cells, differentiation day 7 embryoid bodies (EB), or fibroblasts (T4) (row 1). After 24 h, radioactivity was measured as counts per minute (cpm) from a total cell lysate using a scintillation counter (row 2). Knowing the cellular volume and the number of total cells in each assay (rows 3 and 4), we calculated the total volume of cells (row 5) into which X1 molecule was diluted (cpm μl−1) (row 6). The cpm μl−1 value was interpolated to a standard curve (sheet 2) constructed with increasing concentrations of the tritiated molecule: [cpm μl−1] = 33.954 × [nM molecule] − 898.7. The concentration factor could then be derived by comparing the concentration of the molecule found in cells (row 7) with the original amount added to the culture (row 1).

Supplementary Table 4

Further demonstration of selectivity of X1 for Xist and X inactivation. List of autosomal genes where relative changes in RNA expression are >2 or <−0.5, and relative changes in H3K27me3 enrichment are >4 or <−0.25. All values are logarithmic (base 2).

Supplementary Table 5

List of upregulated genes on the Xi following X1 treatment. Left, all active genes encoded in the X chromosome with FKPM > 0.5 are listed. Right, genes encoded on Xmus, up-regulated with X1. Results are expressed as the relative change (%) in the fraction of reads transcribed from the mus allele with respect to the overall (mus+cas) expression.

Supplementary Video 1

C13 (dominant) conformation of RepA RNA.

Supplementary Video 2

C6′ conformation of RepA RNA with X1 molecule.

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Aguilar, R., Spencer, K.B., Kesner, B. et al. Targeting Xist with compounds that disrupt RNA structure and X inactivation. Nature 604, 160–166 (2022).

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