XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1
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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.
KeywordsGreen Fluorescent Protein Nuclear Retention Export Factor Green Fluorescent Protein mRNA Green Fluorescent Protein Sequence
We are grateful to Joan Steitz for providing reagents and for sharing unpublished data.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.
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