FMRP targets distinct mRNA sequence elements to regulate protein expression

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.

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 2. 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).

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.

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).

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).

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.