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FMRP targets distinct mRNA sequence elements to regulate protein expression

Nature volume 492, pages 382–386 (2012)Cite this article

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Abstract

Fragile X syndrome (FXS) is a multi-organ disease that leads to mental retardation, macro-orchidism in males and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASDs). FXS is typically caused by the loss of fragile X mental retardation 1 (FMR1) expression, which codes for the RNA-binding protein FMRP. Here we report the discovery of distinct RNA-recognition elements that correspond to the two independent RNA-binding domains of FMRP, in addition to the binding sites within the messenger RNA targets for wild-type and I304N mutant FMRP isoforms and the FMRP paralogues FXR1P and FXR2P (also known as FXR1 and FXR2). RNA-recognition-element frequency, ratio and distribution determine target mRNA association with FMRP. Among highly enriched targets, we identify many genes involved in ASD and show that FMRP affects their protein levels in human cell culture, mouse ovaries and human brain. Notably, we discovered that these targets are also dysregulated in Fmr1−/− mouse ovaries showing signs of premature follicular overdevelopment. These results indicate that FMRP targets share signalling pathways across different cellular contexts. As the importance of signalling pathways in both FXS and ASD is becoming increasingly apparent, our results provide a ranked list of genes as basis for the pursuit of new therapeutic targets for these neurological disorders.

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Figure 1: PAR-CLIP of FMR1-family proteins.
Figure 2: Analysis of FMR1-family protein mRNA-binding sites.
Figure 3: RNA-binding assays using natural FMRP target sites containing ACUK and WGGA RREs, and the effect of a KH2 mutation to its target RNA spectrum.
Figure 4: RRE-dependent enrichment criteria for FMRP association with mRNAs.
Figure 5: Ovarian phenotype in Fmr1 −/ − mice.

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Gene Expression Omnibus

Data deposits

Data sets have been submitted to gene expressionomnibus (GEO) under the accession code GSE39686

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Acknowledgements

Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore. We would like to thank the following members of the Tuschl laboratory for their support and assistance: G. Wardle, N. Renwick and I. Ben-Dov. We would like to thank J. Keene for his advice throughout the project. We would like to acknowledge M. Khorshid, L. Burger and M. Zavolan for analysing PAR-CLIP data at the initial stages of the project and discussions. We would like to thank the Memorial Sloan-Kettering Cancer Center in-situ core for their assistance with the mouse histology. Finally, we would like to express our appreciation to all members of the Tuschl laboratory for their assistance and collegiality. This work was supported, in part, by the following agencies: NIH/NCRR/RU CCTS (M.A., UL1RR024143), NSF (U.O., MCB-0822033), Simons Foundation Autism Research Initiative (T.T., CEN5300891) and NIH (T.T., R01 MH080442; Z.W., K08 HD068546).

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  1. Neelanjan Mukherjee & Uwe Ohler

    Present address: Present address: The Berlin Institute for Medical Systems Biology, Max Delbrück Center, 13125 Berlin-Buch, Germany (N.M. and U.O.).,

Authors and Affiliations

  1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, 10065, New York, USA

    Manuel Ascano, Pradeep Bandaru, Jason B. Miller, Jeffrey D. Nusbaum, Mathias Munschauer, Markus Hafner, Zev Williams & Thomas Tuschl

  2. Institute for Genome Sciences and Policy, Duke University, Durham, 27708, North Carolina, USA

    Neelanjan Mukherjee, David L. Corcoran & Uwe Ohler

  3. Department of Obstetrics & Gynecology and Women’s Health, Program for Early and Recurrent Pregnancy Loss, Albert Einstein College of Medicine, Bronx, 10461, New York, USA

    Christine Langlois & Zev Williams

  4. Genomics Resource Center, The Rockefeller University, New York, 10065, New York, USA

    Scott Dewell

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Contributions

M.A. designed, executed, supervised and interpreted experiments. N.M., P.B. and D.L.C. carried out the sequence alignment, annotation and PARalyzer pipeline. N.M. and P.B. performed the computational analysis on the RIP-chip. M.A., J.B.M. and J.D.N. purified FMRP proteins, performed the EMSAs and carried out the quantitative western blots and analyses. M.A. and M.M. performed the RIP-chips. S.D. assisted in the Illumina sequencing of all PAR-CLIP libraries. M.H. helped in the initial PAR-CLIP experiments. C.L. and Z.W. carried out and analysed mouse experiments. U.O. supervised computational efforts. T.T. supervised and helped in the design of experiments. M.A. and T.T. wrote the manuscript.

Corresponding authors

Correspondence to Uwe Ohler or Thomas Tuschl.

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Competing interests

T.T. is co-founder and scientific advisor to Alnylam Pharmaceuticals and Regulus Therapeutics.

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Ascano, M., Mukherjee, N., Bandaru, P. et al. FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature 492, 382–386 (2012). https://doi.org/10.1038/nature11737

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