FMRP targets distinct mRNA sequence elements to regulate protein expression

Journal name:
Nature
Volume:
492,
Pages:
382–386
Date published:
DOI:
doi:10.1038/nature11737
Received
Accepted
Published online

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.

At a glance

Figures

  1. PAR-CLIP of FMR1-family proteins.
    Figure 1: PAR-CLIP of FMR1-family proteins.

    a, FMR1-family proteins comprise two type-I KH domains (cyan). FMRP isoforms (iso) 1 and 7 vary by the presence of exon 12 (black) within KH2. The I304N mutation (red asterisk) is located within the KH2 domain. The arginine–glycine-rich region (RG; orange bars) is also implicated in RNA binding. The lengths of proteins in amino acids are indicated. We established stable inducible cell lines expressing Flag-HA epitope-tagged wild-type and I304N mutants of FMRP (isoforms 1 and 7), and its paralogues FXR1P and FXR2P (ref. 47). b, RNA–FMRP crosslinking comparing CLIP (254nm) to 4SU or 6-thioguanosine (6SG) PAR-CLIP (365nm). RNA-radiolabelled Flag immunoprecipitates of lysates from crosslinked HEK293 cells expressing Flag-HA-tagged FMRP isoform 7 were separated by SDS–polyacrylamide gel electrophoresis (PAGE). The migrations of protein mass standards are indicated. Enrichment of radiolabelled RNA covalently bound to Flag-HA–FMRP (arrow) was determined after normalizing by western blot analysis (not shown). c, 4SU PAR-CLIP of FMR1-family proteins analogous to b.

  2. Analysis of FMR1-family protein mRNA-binding sites.
    Figure 2: Analysis of FMR1-family protein mRNA-binding sites.

    a, Distribution of binding sites within mRNA targets of the FMR1 protein family. b, Two key RREs were inferred from FMRP isoform 1 and 7 binding sites: top, ACUK; bottom, WGGA. c, Distribution of FMRP binding sites, colour-coded on the basis of cERMIT-inferred RREs, across representative targets. Open boxes and thick lines indicate CDS and UTRs, respectively. Numbers indicate nucleotide number. RPKM, reads per kilobase of mature transcript per million mapped reads.

  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 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.

    a, Top left, EMSAs of RNAs representing UBE3A or PPP2CA binding sites containing various RREs. Top right, binding curves and dissociation constants (Kd) are shown. Bottom, the sequences of the RNAs are provided, with WGGA (yellow) and ACUK RREs (cyan) highlighted. b, Effect of the KH2 mutation in FMRP on target sites containing ACUK versus WGGA RREs. The RNA affinities of wild-type and I304N FMRP isoform 1 were compared using binding sites in NF1 (ACUK) and FMR1 (WGGA). c, Binding curves of wild-type and I304N FMRP for an RNA segment representing a mixed RRE binding site in NF1, and several mutant sequence versions (ACUK(−), WGGA(−) and ACUK, WGGA(−)). d, Comparison of FMRP isoform 1 affinity for RRE type in EMSAs and FMRP isoforms 1 and 7 wild-type and I304N PAR-CLIP libraries. Error bars in EMSA summary (left) represent s.d., n = 9 (ACUK) or 8 (WGGA). The ratio of sequence reads aligned to each RRE binding site was calculated between wild-type and I304N FMRP PAR-CLIP libraries. The average sequence-depth ratio of wild-type over I304N binding site, per RRE type, are shown. Error bars in the read-depth analyses (middle and right) represent the average minimum and maximum values across all sub-sampled mutant libraries (n = 14 and 26 for isoforms 1 and 7, respectively).

  4. RRE-dependent enrichment criteria for FMRP association with mRNAs.
    Figure 4: RRE-dependent enrichment criteria for FMRP association with mRNAs.

    ad, Cumulative distribution fraction plots of FMRP targets on the basis of indicated criteria. Transcripts were grouped and colour-coded on the basis of indicated bins. Non-targets are mRNA transcripts with zero PAR-CLIP binding sites, although detectable in the array; total denotes the sum of non-targets and PAR-CLIP-identified targets detectable by RIP-chip. RIP-chip experiments were performed using Flag-HA–FMRP isoform 1 (a). Enrichment of 93 PAR-CLIP identified ASD-related target genes (d). e, Immunoblot densitometry analysis of top-ranking FMRP targets from RIP-chip and PAR-CLIP analyses in HEK293 cells and human brain. In cell culture, target-protein expression differences of indicated proteins were determined upon induction of FMRP isoform 1 or 7 expression. Similarly, relative protein expression was measured using lysates prepared from indicated brain regions of four FXS patients, compared to age/sex/anatomic-matched controls. Error bars denote s.e.m.; n = 2–11 (depending on protein measured and whether the sample was HEK293 or brain lysate). PABPC1 protein level served as a loading and ratio control as it was a gene with PAR-CLIP binding sites but showed no RIP-chip enrichment (−0.10 log2-fold enrichment). *P<0.05.

  5. Ovarian phenotype in Fmr1-/- mice.
    Figure 5: Ovarian phenotype in Fmr1−/ mice.

    ac, Ovaries from wild-type and Fmr1−/− female mice were collected at 3, 9, 12 and 18 weeks and processed for histological (a), morphological (b) and quantitative western analyses (c). By 3 weeks of age, histological staining (haematoxylin) of sectioned ovaries showed a greater than expected number of follicles compared to wild type (a). Ovaries from 18-week-old Fmr1−/− mice are larger than those from wild type and exhibit prominent cysts consistent with corpus luteal development (b). Lysates were prepared from 9-, 12- and 18-week-old ovaries from two different wild-type and knockout mice each, and analysed by quantitative western blot using Mtor, Sash1 and Tsc2 antibodies. As for human samples, Pabpc1 was used for normalization (c).

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Gene Expression Omnibus

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Author information

Affiliations

  1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, 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, North Carolina 27708, USA

    • Neelanjan Mukherjee,
    • David L. Corcoran &
    • Uwe Ohler
  3. Program for Early and Recurrent Pregnancy Loss, Department of Obstetrics & Gynecology and Women’s Health, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    • Christine Langlois &
    • Zev Williams
  4. Genomics Resource Center, The Rockefeller University, New York, New York 10065, USA

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

    • Neelanjan Mukherjee &
    • Uwe Ohler

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.

Competing financial interests

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

Corresponding authors

Correspondence to:

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

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Comments

  1. Report this comment #53056

    Manuel Ascano, Jr. said:

    The catalytic alpha-subunit of PI3 kinase (Pik3ca) was mistakenly referred to as the gene previously identified target (in references 42 and 43), when it was actually the beta-subunit (Pik3cb). In our study, the mRNAs for both human catalytic subunits of PI3 kinase (PIK3CA and PIK3CB) were identified as targets of FMRP.

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