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FMR1 targets distinct mRNA sequence elements to regulate
protein expression
Manuel Ascano Jr.1, Neelanjan Mukherjee2,5, Pradeep Bandaru1, Jason B. Miller1, Jeff
Nusbaum, David L. Corcoran2, Christine Langlois3, Mathias Munschauer, Scott Dewell4,
Markus Hafner1, Zev Williams1,3, Uwe Ohler2,5,*, and Thomas Tuschl1,*
1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller
University, New York, NY 10065, USA
2Institute for Genome Sciences and Policy, Duke University, Durham, NC 27708, USA
3Program for Early and Recurrent Pregnancy Loss, Department of Obstetrics & Gynecology and
Women's Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA
4Genomics Resource Center, The Rockefeller University, New York, NY, 10065, USA
Abstract
Fragile-X Syndrome (FXS) is a multi-organ disease leading 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 (ASD). FXS is typically
caused by the loss of FRAGILE X-MENTAL RETARDATION 1 (FMR1) expression, which
encodes for the RNA-binding protein (RBP), FMRP. We report the discovery of distinct RNA
recognition elements (RREs) that correspond to the two independent RNA binding domains of
FMRP, and the binding sites within the mRNA targets for wild-type and I304N mutant FMRP
isoforms and its paralogs, FXR1 and FXR2. RRE frequency, ratio, and distribution determine
target mRNA association with FMRP. Among highly-enriched targets, we identified many genes
involved in ASD and demonstrate that FMRP affects their protein levels in cell culture, mice, and
human brain. Unexpectedly, we discovered that these targets are also dysregulated in Fmr1-/-
mouse ovaries, showing signs of premature follicular overdevelopment. These results indicate that
FMRP targets shared signaling pathways across different cellular contexts. As it is become
increasingly appreciated that signaling pathways are important to FXS and ASD, our results here
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*Corresponding authors Thomas Tuschl ttuschl@rockefeller.edu.
5Present address: The Berlin Institute for Medical Systems Biology, Max Delbrück Center, Berlin-Buch, Germany
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.N.
purified FMRP proteins, performed the EMSAs, and carried out the quantitative Westerns 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 analyzed 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 INTEREST
T.T. is co-founder and scientific advisor to Alnylam Pharmaceuticals and Regulus Therapeutics.
HHS Public Access
Author manuscript
Nature. Author manuscript; available in PMC 2013 June 20.
Published in final edited form as:
Nature. 2012 December 20; 492(7429): 382–386. doi:10.1038/nature11737.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
provide a molecular guide towards the pursuit of novel therapeutic targets for these neurological
disorders.
INTRODUCTION
Most clinical cases of FXS are a result of a hyper-expansion and methylation of CGG
repeats within the promoter of FMR1, leading to a loss of its expression1-3. The FMR1 RBP
family has three members, FMR1, FXR1, and FXR2, which possess two centrally located
KH domains and a C-terminal arginineglycine (RG)-rich region implicated in mRNA
binding 4-7. FMR1 encodes for multiple protein isoforms, but is predominantly expressed as
a 69 kDa protein (isoform 7)8,9. Isoform (iso) 1 and six other alternative splice variants
include exon 12, with iso1 encoding the full-length protein (71 kDa). Exon 12 insertion
lengthens the second KH (KH2) RBD, possibly influencing FMR1 RNA-binding specificity
or affinity. The I304N mutation, described in a FXS patient, is also located in the KH2 and
is reported to attenuate association with RNA and polysomes10-12. FMR1 proteins are
implicated in various RNA processes including RNA subcellular localization by facilitating
nucleo-cytoplasmic shuttling13 and association with motor proteins14-16. FMR1 proteins
were also suggested to mediate translational regulation12,17. Given the critical role of FMR1
in human cognition and premature ovarian insufficiency18,19, intensive efforts towards the
identification of its RNA targets have been employed, with the goal that their discovery
would shed light upon the array of related disorders and provide options for molecular
therapy18-26. No precise RRE has been defined and very few bona fide mRNA targets are
confirmed27.
RESULTS AND DISCUSSION
To identify the binding sites of FMR1 family proteins (Fig. 1a and Supplementary Fig. 1),
we first compared photocrosslinking methods28-30 using stable FLAG-HA FMR1 iso7
expressing HEK293 cells (Fig. 1b), as these cells and human brain share 90% of expressed
genes based on a comparison of existing RNAseq datasets31-33 (Supplementary Fig. 2). The
difference in FMR1 levels between the experimental system and brain is 1.3 fold, as
calculated using RNAseq data and the quantitated expression of FMR1 in our stable cells.
We found that 4SU PAR-CLIP provided the highest yield of crosslinked RNAs, and used
this approach for all FMR1 family proteins (Fig. 1c). cDNA libraries were generated after
PAR-CLIP and Illumina-sequenced (Supplementary Table 1). Genome-aligned reads were
grouped by PARalyzer34 to identify segments of RNA that represented peaks of T-to-C
conversion, termed binding sites. PARalyzer separated closely spaced binding sites
connected by overlapping reads and yielded a median RNA segment length of 33 nt
(Supplementary Fig. 3). FMRP iso1 and 7 bound to approx. 80,000-100,000 sites, of which
> 85% mapped to ~6,000 mRNAs (Supplementary Tables 1, 2, and https://
fmrp.rockefeller.edu). FXR1 and FXR2 protein binding sites are comprised within FMRP
binding sites with an overlap of 95% (Supplementary Table 3).
Nearly all mRNA binding sites were located in exons (>90%) (Fig. 2a) and distributed
between CDS and 3'UTR (>95%, total) with slightly more CDS sites, similar to distributions
seen for other cytoplasmic RBPs28. The computational sequence analysis method cERMIT35
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revealed two major RREs, ACUK and WGGA (K=G/U, and W=A/U) (Fig 2b,
Supplementary Fig. 4). Together, ACUK and WGGA RREs were found in ≥50% of mRNA
binding sites in iso1 and iso7; they occurred exclusively or together within the same binding
site (Fig. 2c). Remaining binding sites typically contained close derivatives of either RRE.
We performed electrophoretic mobility shift assays (EMSAs) to test RNAs representing
FMRP binding sites using recombinant FMRP purified from Sf9 cells (Supplementary Fig. 5
and 6). FMRP target sites were selected based on whether they contained ACUK, WGGA or
Mixed (ACUK/WGGA) RREs (Supplementary Table 4). Testing RNAs of various lengths,
we found that oligonucleotide lengths of ≥45 nt were required to observe gel shifts and reach
dissociation constants below 0.5 μM, suggesting RNA backbone contacts outside the RREs
contribute to the association in vitro. WGGA-containing RNAs exhibited the widest range
and strongest affinities, generally correlating with the number of RREs within a PARalyzer-
defined binding site. An RNA segment containing nine WGGAs bound almost 2 orders of
magnitude tighter than those containing one WGGA, whereas binding of ACUK-containing
RNAs varied only 5-fold. EMSAs using RNAs representing target sites within PPP2CA, and
UBE3A are shown (Fig. 3a).
We also tested sites in APP and FMR1 mRNA (Supplementary Fig. 7), two previously
identified mRNA targets. APP was originally discovered as an FMRP target based on a
predicted G-quartet region7 although the actual site was subsequently identified in vitro at an
upstream segment36, which was identified here (APP Site 1) as containing WGGA. FMRP
targets its own mRNA though it was an association only observed in vitro5,6,37 and in
immunoprecipitates38.
Recombinant I304N iso1 FMRP showed a ~10-fold average decrease in its affinity towards
ACUK-containing RNAs compared to wt FMRP iso1 (Fig. 3b and Supplementary Table 5).
We characterized binding to wt and mutant RNA sequences derived from an NF1 mixed
RRE site (Fig. 3c). I304N FMRP bound wt and NF1 ACUK(-) RNAs similarly, whereas wt
FMRP showed a two-fold reduction in affinity for NF1 ACUK(-) RNA. Additionally, wt
FMRP bound NF1 ACUK, WGGA(-) RNA similar to I304N FMRP for NF1 WGGA(-)
RNA. Our results indicate that the KH2 domain associates with the ACUK RRE. Since
FMRP associates with mRNAs at more than one binding site, its association at multiple sites
will impact the final regulatory effect. We compared the distribution of RREs in I304N
FMRP vs. wt FMRP PAR-CLIPs and found a transcriptome-wide depletion in the recovery
of sequence reads for ACUK binding sites for both I304N isoforms, consistent with the
biochemistry (Fig. 3d). Interestingly, the RRE fractional distribution of I304N FMRP iso1
was similar to wt FMRP iso7, suggesting that alteration of the KH2 domain by mutation or
exon insertion affects binding-site selectivity. Taken together, the biochemical and PAR-
CLIP results with I304N FMRP indicate that we identified the natural target sites of the
FMRP KH2 domain.
To rank FMRP targets, we measured their enrichment in RIP-chip, which would indicate
stable interactions. 3,593 PAR-CLIP-identified targets showed enrichment by RIP-chip, of
which 939 genes were two- to six-fold enriched; 646 transcripts were two-fold enriched but
not identified as PAR-CLIP targets. We used binding-site information obtained by PAR-
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CLIP to infer the salient features for stable association in RIP-Chip (Fig. 4, Supplementary
Fig. 8, and Supplementary Table 6). Increasing frequency of WGGA- and ACUK-
containing elements led to greater RIP-chip enrichment, in agreement with in vitro affinity
measurements. On average, top targets contained more RRE binding sites (18 per transcript)
compared to the least-enriched targets (13 per transcript). Top-ranked targets had a CDS:
3'UTR binding site ratio of 3.7:1 compared to bottom-ranked targets that had 1:2.
Transcripts with ACUK:WGGA RRE ratios <1 were the most significantly enriched
population. Importantly, we identified 93 genes independently implicated in ASD among the
highly-enriched FMRP targets (Fig. 4d and Supplementary Table 7), which is striking given
the relationship of FXS with ASD20,21,39.
Enrichment of RNAs in RIP-chip depends on the saturation of target sites with FMRP. To
estimate the yield of saturation, protein copy number, target mRNA copies, and the number
of binding sites within those transcripts have to be accounted. We determined endogenous
and co-expressed FLAG-HA-tagged FMR1 protein copy numbers to be approx. 60,000 and
70,000 molecules per cell, respectively, using quantitative Western blotting and reference
recombinant protein. Each cell contains 20 pg total RNA, of which 4% are the approx. 1
Mio mRNA molecules/cell. Considering their relative abundance based on HEK293
RNAseq, we estimate that an equal distribution of FMRP would occupy 6% of binding sites/
cell among its 6,000 target transcripts, or up to 20% if FXR1 and 2 protein estimates are
included. Since PAR-CLIP-identified targets had varying enrichment, the association of
FMRP with them is not uniform. The ~3,500 transcripts enriched in RIP-chip are estimated
to have at least 18% FMRP binding site occupancy, whereas top-ranked 900 genes (two-fold
enriched) potentially exhibit 78%. The presence of multiple binding sites within targets
suggests that multiple FMR1 family proteins bind each transcript to influence their
regulation.
To assess the impact of FMR1 binding sites on mRNA stability, siRNA knockdown of
FMR1 or the FMR1 family was performed and mRNA expression profiles were analysed by
microarray. We found no evidence for FMR1 affecting target mRNA abundance (data not
shown).
A panel of FMR1-targets were selected based on enrichment in RIP-Chip, low-to-
intermediate expression in RNAseq, similar abundance in human brain (using published
microarray31 and RNAseq datasets32,33) and with documented neurological and human
disease relevance, then analysed them by quantitative Western blot to determine their
protein levels as a function of FMRP expression (Fig. 4e). FXR2, HUWE1, KDM5C, and
MTOR protein levels, among others, showed up to 30% reduction in protein levels upon
expression of FMR1, in HEK293. We analysed lysates prepared from human postmortem
brains. Four FXS brains (Supplementary Fig. 9) were available with age/sex/anatomic-
matched controls from pre-frontal cortical, hippocampus, and cerebellar regions. While only
four of eight antibodies yielded quantifiable bands in brain lysates, we observed a general
trend of elevated target protein expression levels in FXS brains. This is the inverse of FMR1
overexpression effects in HEK293, and consistent with FMRP affecting the protein levels of
its mRNA targets.
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The mRNA targets identified here are from a human transcriptome where the vast majority
of genes are comparably expressed in human brain (Supplementary Fig. 2). We discovered
ASD-related and numerous other genes implicated in neuronal disorders associated with
FXS and validated representatives by EMSA, RIP-chip, and immunoblot. We found genes
involved in Angelman (AS), Prader-Willi, Rett, and Cornelia de Lange Syndromes.
Interestingly, the ASD and AS-associated gene UBE3A ubiquitinates ARC and SACS140;
ARC is a well-known target and here we identify SACS1 as a targeted transcript. These
findings potentially provide the molecular link to tie together elements of clinically
overlapping disorders, principally setting a molecular target framework for characterizing
the connections between FXS and its associated phenotypes.
Although FX-related diseases are primarily considered CNS disorders, at least two other
target organs are affected, the testes and ovaries. We reasoned that changes in FMR1
expression lead to dysregulation of largely overlapping sets of targets shared across all
affected organs. Thus dysregulated genes and pathways in brain might also contribute to
phenotypes in testes and ovary. We therefore examined the ovaries of Fmr1-/- mice41 since
CNS and macroorchidism phenotypes were reported, yet ovary development had largely
been under-investigated. Ovaries from Fmr1-/- mice were markedly larger by 3 weeks post-
birth compared to wt controls (Fig. 5a-b). At 12 and 18 wks post-birth, KO ovaries were
22% and 72% larger by mass compared to age-matched controls, respectively. Importantly,
we found increased protein levels of Tsc2, Sash1 and Mtor (Fig. 5c). As it is independently
known that the Mtor pathway can regulate ovarian development, it is tempting to conclude
that increased activity, in the absence Fmr1 expression, contributes to enlarged ovaries
histologically consistent with precocious follicular development. These observations suggest
that Fmr1-KO mice have the potential to model FXS-related premature ovarian
insufficiency in which it remains unclear whether elevated FMR1 mRNA or the observed
reduction of FMRP protein itself is causative in female carriers affected by this disease.
Elevated signal transduction activity of the Mtor pathway42,43 was reported in Fmr1 KO-
mice, and was attributed to Fmrp targeting of Pik3ca mRNA. Indeed, PAR-CLIP identified
several FMRP binding sites within the PIK3CA transcript. However, we find that it is a less-
enriched RIP-chip target compared to MTOR and TSC2, whose protein levels appear
regulated in an FMRP-dependent manner. Interestingly, recent evidence demonstrated that
Tsc mutant mice44-46, which have increased mTOR activity, had impaired mGluR-LTD and
protein synthesis compared to Fmr1-/- mice; crossing Tsc2+/- with Fmr1-/y mice corrected
the phenotypes44. Given our results we suspect that in Tsc2+/-, Fmr1-/y mice, Tsc2 and Mtor
protein levels (among others) were elevated, correcting the balance of protein expression
and leading to the reversal of phenotypes observed. The reported model44 by which separate
pools of mRNA are differentially regulated by partially convergent pathways in FMRP
(in)dependent ways, remains unclear. This is in part because FMRP associates with
transcripts of ERK pathway components as well. Therapeutic targeting of the MTOR
pathway has become an important goal – but must be further guided by additional functional
analysis, particularly of FMRP targets upstream and downstream of MTOR and
interconnected signaling pathways (Supplementary Fig. 10). Combined, our validation work
in Fmr1-KO mouse ovaries and in human brain demonstrate that the effect of FMRP
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binding to specific target genes identified in cell culture is extensible to physiologically
relevant contexts.
METHODS SUMMARY
Methods are described in greater detail within Supplementary Information. FVB129P2
Fragile-X mice were a kind gift from Dr. Suzanne Zukin (Albert Einstein College of
Medicine). Gateway plasmids (Invitrogen) generated in this study will be deposited in
addgene.org. FlpIn T-Rex HEK293 (Invitrogen) inducible-stable cell lines were generated
per manufacturer's instrutions. The titers, source and use of antibodies used in this study are
listed in Supplementary Information. PAR-CLIP was performed essentially as described,
except that the second RNase T1 digestion was omitted following the IP. Recombinant wt
and mutant FMRP iso1 proteins were purified using baculoviral expression system
(Invitrogen). Electrophoretic mobility shift assays and Western blots were quantified using
ImageGauge (Fuji). Parameters of computation analyses are described in Supplementary
Information and within the relevant sections within https://fmrp.rockefeller.edu/. Relevant
datasets, including raw data are available at https://fmrp.rockefeller.edu/ and GEO
(GSE39686).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGEMENTS
Human tissue was obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University
of Maryland, Baltimore, MD. We would like to thank the following members of the Tuschl lab for their support and
assistance: Greg Wardle, Dr. Neil Renwick, and Dr. Iddo Ben-Dov. We would like to thank Dr. Jack Keene for his
invaluable advice throughout the project. We would like to acknowledge Dr. Mohsen Khorshid, Dr. Lukas Burger,
and Dr. Mihaela Zavolan for analyzing PAR-CLIP data at the initial stages of the project and discussions. We
would like to thank the MSKCC in-situ core for their assistance with the mouse histology. Finally, we would like to
express our appreciation for 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), Simons
Foundation Autism Research Initiative (T.T., CEN5300891) and NIH (T.T., R01 MH080442).
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Figure 1. PAR-CLIP of FMR1 family proteins
a, FMR1 family proteins comprise two type-I KH domains (cyan). FMRP isoform 1 and 7
(iso1 and 7) vary by the presence of exon 12 (black) within KH2. The I304N mutation is
located within the KH2 domain (red asterisk). The RG-rich region (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 wt and I304N
mutants of FMR1 (iso1 and 7), and its homologs FXR1 and 247. b, RNA-FMRP
crosslinking comparing CLIP (254 nm) to 4SU-or 6SG-PAR-CLIP (365 nm). RNA-
radiolabeled FLAG immunoprecipitates (IPs) of lysates from crosslinked FLAG-HA-
FMRP-iso7-expressing HEK293 cells were separated by SDS-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).
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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
major RREs were inferred from FMRP iso1 and iso7 binding sites. c, Distribution of FMRP
binding sites, color-coded based on cERMIT-inferred RREs, across representative targets.
Open boxes and thick lines indicate CDS and UTRs, respectively; numbers indicate
nucleotide number.
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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, EMSAs of RNAs representing UBE3A or PPP2CA binding sites containing various
RREs. Binding curves and constants are shown. The sequences of the RNAs are provided
with WGGA (yellow) and ACUK RREs (cyan) highlighted. b, Impact of KH2 mutation in
FMRP on target sites containing ACUK versus WGGA RREs. The RNA affinities of wt and
I304N FMRP iso1 were compared using binding sites in NF1 (ACUK) and FMR1 (WGGA).
c, Binding curves of wt 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 iso1 affinity for RRE type in EMSAs and
FMRP iso1 and 7 wt and I304N PAR-CLIP libraries. Error bars in EMSA summary
represent s.d., n= 9 (ACUK), or 8 (WGGA). The ratio of sequence reads aligned to each
RRE binding site was calculated between wt and I304N FMRP PAR-CLIP libraries. The
average sequence-depth ratio of wt over I304N binding site, per RRE-type, are shown. Error
bars in the read-depth analyses represent the avg. min and max values across all subsampled
mutant libraries (n= 14 and 26 for iso 1 and 7, respectively).
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Figure 4. RRE-dependent enrichment criteria for FMRP association with mRNAs
a, RIP-chip experiments were performed using FLAG-HA-FMRP iso1. a-d, Cumulative
distribution fraction plots of FMRP targets based on indicated criteria. Transcripts were
grouped and color-coded based on indicated bins. Non-targets are mRNA transcripts with
zero PAR-CLIP binding sites, although detectable in the array; total is the sum of non-
targets and PAR-CLIP identified targets detectable by RIP-chip. d, Enrichment of ninety-
three PAR-CLIP identified ASD-related target genes. e, Immunoblot densitometry analysis
of top-ranking FMRP targets from RIP-chip and PAR-CLIP analyses in HEK293 and human
brain. In cell culture, target protein expression differences of indicated proteins were
determined upon induction of FMR1 iso1 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 represent s.e.m., with n
= 2-11 (depending on protein measured and whether the sample was HEK293 or brain
lysate). PABPC1 protein level served as loading and ratio control as it was a gene with
PAR-CLIP binding sites but showed no RIP-chip enrichment (-0.10 LFE).
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Figure 5. Ovarian phenotype in Fmr1-/- mice
Ovaries from wt and Fmr1-/- female mice were harvested at 3, 9, 12, and 18 wks and
processed for histological (a), morphological (b), and quantitative western analyses (c). a,
By 3 wks of age, histological staining (hematoxylin) of sectioned ovaries show greater than
expected number of follicles compared to wt. b, Ovaries from 18 wk old Fmr1-/- mice are
larger than wt and exhibit prominent cysts consistent with corpus luteal development. c,
Lysates were prepared from 9, 12, and 18 wk ovaries from two different wt and KO mice
each, and analysed by quantitative Western using Mtor, Sash1, and Tsc2 antibodies. As in
human samples, Pabpc1 was used for normalization.
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