Advertisement

Chromosoma

, Volume 116, Issue 4, pp 373–383 | Cite as

XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1

  • Hannah R. Cohen
  • Barbara PanningEmail author
Research Article

Abstract

During splicing and polyadenylation, factors that stimulate export from the nucleus are recruited to nascent mRNAs. X-inactive specific transcript (XIST) RNA is unusual among capped, spliced, polyadenylated transcripts in that it accumulates exclusively in the nucleus. It is well established that, at steady state levels, XIST RNA is primarily nuclear. However, it was unknown whether XIST RNA spends its entire lifetime in the nucleus (nuclear retention) or passes briefly through the cytoplasm during maturation, like many other functional RNAs. In this study, we present the first evidence that XIST RNA exhibits nuclear retention. We report that a green fluorescent protein (GFP)–XIST fusion RNA is detected in the nucleus and not the cytoplasm, and GFP is not translated. XIST RNA does not shuttle in a heterokaryon assay or move between chromosomes in the same nucleus when expressed at wild-type levels. These results indicate that XIST RNA’s nuclear localization is mediated by nuclear retention rather than export followed by import. We present evidence that the export factor TAP/NXF1 binds poorly to XIST RNA in comparison to exported mRNAs, suggesting that reduced TAP/NFX1 binding may contribute to nuclear retention of XIST RNA.

Keywords

Green Fluorescent Protein Nuclear Retention Export Factor Green Fluorescent Protein mRNA Green Fluorescent Protein Sequence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We are grateful to Joan Steitz for providing reagents and for sharing unpublished data. We appreciate the advice and technical help of Yingqun Huang. Sincere thanks go to Christine Guthrie and to all the members of the Panning lab for critical reading of this manuscript. We thank Angela Anderson for cell line construction, Jocelyn Turner for help with flow cytometry analysis, and Yu-Tsueng Liu for advice on making heterokaryons. We are deeply grateful to Judith Sharp for help with experiments and writing this manuscript. HRC is a recipient of a National Science Foundation predoctoral fellowship. BP is a Pew Scholar and is funded by the National Institutes of Health and the Sandler Family Foundation.

Supplementary material

References

  1. Andersen AA, Panning B (2003) Epigenetic gene regulation by noncoding RNAs. Curr Opin Cell Biol 15:281–289PubMedCrossRefGoogle Scholar
  2. Andrulis ED, Werner J, Nazarian A, Erdjument-Bromage H, Tempst P, Lis JT (2002) The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature 420:837–841PubMedCrossRefGoogle Scholar
  3. Berk AJ, Sharp PA (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721–732PubMedCrossRefGoogle Scholar
  4. Bernstein E, Allis CD (2005) RNA meets chromatin. Genes Dev 19:1635–1655PubMedCrossRefGoogle Scholar
  5. Brockdorff N, Ashworth A, Kay GF, McCabe VM, Norris DP, Cooper PJ, Swift S, Rastan S (1992) The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell 71:515–526PubMedCrossRefGoogle Scholar
  6. Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R, Willard HF (1991) A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349:38–44PubMedCrossRefGoogle Scholar
  7. Brown CJ, Hendrich BD, Rupert JL, Lafreniere RG, Xing Y, Lawrence J, Willard HF (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71:527–542PubMedCrossRefGoogle Scholar
  8. Buzin CH, Mann JR, Singer-Sam J (1994) Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies. Development 120:3529–3536PubMedGoogle Scholar
  9. Caceres JF, Screaton GR, Krainer AR (1998) A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm. Genes Dev 12:55–66PubMedGoogle Scholar
  10. Carmo-Fonseca M, Tollervey D, Pepperkok R, Barabino SM, Merdes A, Brunner C, Zamore PD, Green MR, Hurt E, Lamond AI (1991) Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J 10:195–206PubMedGoogle Scholar
  11. Chu F, Nusinow DA, Chalkley RJ, Plath K, Panning B, Burlingame AL (2006) Mapping post-translational modifications of the histone variant macroH2A1 using tandem mass spectrometry. Mol Cell Proteomics 5:194–203PubMedCrossRefGoogle Scholar
  12. Clemson CM, McNeil JA, Willard HF, Lawrence JB (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol 132:259–275PubMedCrossRefGoogle Scholar
  13. Clemson CM, Chow JC, Brown CJ, Lawrence JB (1998) Stabilization and localization of Xist RNA are controlled by separate mechanisms and are not sufficient for X inactivation. J Cell Biol 142:13–23PubMedCrossRefGoogle Scholar
  14. Clerc P, Avner P (1998) Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation. Nat Genet 19:249–253PubMedCrossRefGoogle Scholar
  15. de Napoles M, Mermoud JE, Wakao R, Tang YA, Endoh M, Appanah R, Nesterova TB, Silva J, Otte AP, Vidal M et al (2004) Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev Cell 7:663–676PubMedCrossRefGoogle Scholar
  16. Dower K, Rosbash M (2002) T7 RNA polymerase-directed transcripts are processed in yeast and link 3′ end formation to mRNA nuclear export. RNA 8:686–697PubMedCrossRefGoogle Scholar
  17. Dower K, Kuperwasser N, Merrikh H, Rosbash M (2004) A synthetic A tail rescues yeast nuclear accumulation of a ribozyme-terminated transcript. RNA 10:1888–1899PubMedCrossRefGoogle Scholar
  18. Fang J, Chen T, Chadwick B, Li E, Zhang Y (2004) Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in initiation of X inactivation. J Biol Chem 279:52812–52815PubMedCrossRefGoogle Scholar
  19. Femino AM, Fogarty K, Lifshitz LM, Carrington W, Singer RH (2003) Visualization of single molecules of mRNA in situ. Methods Enzymol 361:245–304PubMedGoogle Scholar
  20. Fok V, Friend K, Steitz JA (2006) Epstein–Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling. J Cell Biol 173:319–325PubMedCrossRefGoogle Scholar
  21. Hall LL, Byron M, Sakai K, Carrel L, Willard HF, Lawrence JB (2002) An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells. Proc Natl Acad Sci USA 99:8677–8682PubMedCrossRefGoogle Scholar
  22. Henry RA, Tews B, Li X, Scott MJ (2001) Recruitment of the male-specific lethal (MSL) dosage compensation complex to an autosomally integrated roX chromatin entry site correlates with an increased expression of an adjacent reporter gene in male Drosophila. J Biol Chem 276:31953–31958PubMedCrossRefGoogle Scholar
  23. Herold A, Teixeira L, Izaurralde E (2003) Genome-wide analysis of nuclear mRNA export pathways in Drosophila. EMBO J 22:2472–2483PubMedCrossRefGoogle Scholar
  24. Hilleren P, McCarthy T, Rosbash M, Parker R, Jensen TH (2001) Quality control of mRNA 3′-end processing is linked to the nuclear exosome. Nature 413:538–542PubMedCrossRefGoogle Scholar
  25. Hong YK, Ontiveros SD, Chen C, Strauss WM (1999) A new structure for the murine Xist gene and its relationship to chromosome choice/counting during X-chromosome inactivation. Proc Natl Acad Sci USA 96:6829–6834PubMedCrossRefGoogle Scholar
  26. Hong YK, Ontiveros SD, Strauss WM (2000) A revision of the human XIST gene organization and structural comparison with mouse Xist. Mamm Genome 11:220–224PubMedCrossRefGoogle Scholar
  27. Huang Y, Gattoni R, Stevenin J, Steitz JA (2003) SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol Cell 11:837–843PubMedCrossRefGoogle Scholar
  28. Huang Y, Yario TA, Steitz JA (2004) A molecular link between SR protein dephosphorylation and mRNA export. Proc Natl Acad Sci USA 101:9666–9670PubMedCrossRefGoogle Scholar
  29. Izaurralde E, Lewis J, Gamberi C, Jarmolowski A, McGuigan C, Mattaj IW (1995) A cap-binding protein complex mediating U snRNA export. Nature 376:709–712PubMedCrossRefGoogle Scholar
  30. Johnson CV, Singer RH, Lawrence JB (1991) Fluorescent detection of nuclear RNA and DNA: implications for genome organization. Methods Cell Biol 35:73–99PubMedCrossRefGoogle Scholar
  31. Kageyama Y, Mengus G, Gilfillan G, Kennedy HG, Stuckenholz C, Kelley RL, Becker PB, Kuroda MI (2001) Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site. EMBO J 20:2236–2245PubMedCrossRefGoogle Scholar
  32. Kelley RL, Meller VH, Gordadze PR, Roman G, Davis RL, Kuroda MI (1999) Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell 98:513–522PubMedCrossRefGoogle Scholar
  33. Lawrence JB, Singer RH (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res 13:1777–1799PubMedCrossRefGoogle Scholar
  34. Lee JT, Jaenisch R (1997) Long-range cis effects of ectopic X-inactivation centres on a mouse autosome. Nature 386:275–279PubMedCrossRefGoogle Scholar
  35. Lyon MF (1961) Gene action in the X-chromosome of the mouse (Mus musculus L) Nature 190:372–373PubMedCrossRefGoogle Scholar
  36. Madore SJ, Wieben ED, Kunkel GR, Pederson T (1984) Precursors of U4 small nuclear RNA. J Cell Biol 99:1140–1144PubMedCrossRefGoogle Scholar
  37. Maquat LE (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA 1:453–465PubMedGoogle Scholar
  38. Meller VH, Gordadze PR, Park Y, Chu X, Stuckenholz C, Kelley RL, Kuroda MI (2000) Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila. Curr Biol 10:136–143PubMedCrossRefGoogle Scholar
  39. Migeon BR, Winter H, Kazi E, Chowdhury AK, Hughes A, Haisley-Royster C, Morrison H, Jeppesen P (2001) Low-copy-number human transgene is recognized as an X inactivation center in mouse ES cells, but fails to induce cis-inactivation in chimeric mice. Genomics 71:156–162PubMedCrossRefGoogle Scholar
  40. Panning B (2004) X inactivation in mouse ES cells: histone modifications and FISH. Methods Enzymol 376:419–428PubMedGoogle Scholar
  41. Park Y, Kelley RL, Oh H, Kuroda MI, Meller VH (2002) Extent of chromatin spreading determined by roX RNA recruitment of MSL proteins. Science 298:1620–1623PubMedCrossRefGoogle Scholar
  42. Plath K, Mlynarczyk-Evans S, Nusinow DA, Panning B (2002) Xist RNA and the mechanism of X chromosome inactivation. Annu Rev Genet 36:233–278PubMedCrossRefGoogle Scholar
  43. Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, de la Cruz CC, Otte AP, Panning B, Zhang Y (2003) Role of histone H3 lysine 27 methylation in X inactivation. Science 300:131–135PubMedCrossRefGoogle Scholar
  44. Politz JC, Tuft RA, Pederson T, Singer RH (1999) Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells. Curr Biol 9:285–291PubMedCrossRefGoogle Scholar
  45. Rodrigues JP, Rode M, Gatfield D, Blencowe BJ, Carmo-Fonseca M, Izaurralde E (2001) REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus. Proc Natl Acad Sci USA 98:1030–1035PubMedCrossRefGoogle Scholar
  46. Seidl CI, Stricker SH, Barlow DP (2006) The imprinted Air ncRNA is an atypical RNAPII transcript that evades splicing and escapes nuclear export. Embo J 25:3565–3575PubMedCrossRefGoogle Scholar
  47. Schwartz RJ, Rothblum KN (1981) Gene switching in myogenesis: differential expression of the chicken actin multigene family. Biochemistry 20:4122–4129PubMedCrossRefGoogle Scholar
  48. Smith KP, Byron M, Clemson CM, Lawrence JB (2004) Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands. Chromosoma 113:324–335PubMedCrossRefGoogle Scholar
  49. Stutz F, Bachi A, Doerks T, Braun IC, Seraphin B, Wilm M, Bork P, Izaurralde E (2000) REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export. RNA 6:638–650PubMedCrossRefGoogle Scholar
  50. Vinciguerra P, Stutz F (2004) mRNA export: an assembly line from genes to nuclear pores. Curr Opin Cell Biol 16:285–292PubMedCrossRefGoogle Scholar
  51. Visa N, Izaurralde E, Ferreira J, Daneholt B, Mattaj IW (1996) A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. J Cell Biol 133:5–14PubMedCrossRefGoogle Scholar
  52. Wutz A, Jaenisch R (2000) A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol Cell 5:695–705PubMedCrossRefGoogle Scholar
  53. Wutz A, Rasmussen TP, Jaenisch R (2002) Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat Genet 30:167–174PubMedCrossRefGoogle Scholar
  54. Xing YG, Lawrence JB (1991) Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol 112:1055– 1063PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Department of Biochemistry and BiophysicsUniversity of California San FranciscoSan FranciscoUSA

Personalised recommendations