Fragile X Mental Retardation Protein FMRP Binds mRNAs in the Nucleus

Abstract

The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grant HD41591-01 from the National Institute of Child Health and Human Development and by the Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International to S.C. M.K. was supported in part by the Neuroscience program. M.B. was supported by an NSF CAREER award.

We thank members of the lab for their thoughtful reading of earlier drafts of the manuscript and Chris Schoenherr, Bill Greenough, and Andy Belmont for providing helpful comments. We also thank Marie Louise Hammarskjold, Lyne Levesque, and Andre Hoogeveen for providing antibodies and constructs; Lin-Feng Chen for the use of his fluorescent microscope; Kannanganattu Prasanth for advice and constructs for the RNase treatment of cells; and Edouard Khandjian for providing the STEK cells. Finally, we thank Erin Patton for input on the beginning stages of this project and for making some of the constructs and reagents used here.