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. 2008 Jan 15;105(2):734-9.
doi: 10.1073/pnas.0710991105. Epub 2008 Jan 9.

On BC1 RNA and the fragile X mental retardation protein

Anna Iacoangeli  1 , Timofey S Rozhdestvensky, Natalia Dolzhanskaya, Barthélémy Tournier, Janin Schütt, Jürgen Brosius, Robert B Denman, Edouard W Khandjian, Stefan Kindler, Henri Tiedge
Affiliations

On BC1 RNA and the fragile X mental retardation protein

Anna Iacoangeli et al. Proc Natl Acad Sci U S A. .

Abstract

The fragile X mental retardation protein (FMRP), the functional absence of which causes fragile X syndrome, is an RNA-binding protein that has been implicated in the regulation of local protein synthesis at the synapse. The mechanism of FMRP's interaction with its target mRNAs, however, has remained controversial. In one model, it has been proposed that BC1 RNA, a small non-protein-coding RNA that localizes to synaptodendritic domains, operates as a requisite adaptor by specifically binding to both FMRP and, via direct base-pairing, to FMRP target mRNAs. Other models posit that FMRP interacts with its target mRNAs directly, i.e., in a BC1-independent manner. Here five laboratories independently set out to test the BC1-FMRP model. We report that specific BC1-FMRP interactions could be documented neither in vitro nor in vivo. Interactions between BC1 RNA and FMRP target mRNAs were determined to be of a nonspecific nature. Significantly, the association of FMRP with bona fide target mRNAs was independent of the presence of BC1 RNA in vivo. The combined experimental evidence is discordant with a proposed scenario in which BC1 RNA acts as a bridge between FMRP and its target mRNAs and rather supports a model in which BC1 RNA and FMRP are translational repressors that operate independently.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Binding of FMRP to BC1 RNA is nonspecific. (A and B) EMSA was performed with 32P-labeled BC1 RNA or N19 RNA and increasing amounts of FMRP as indicated. Monovalent cations were used as follows: 750 mM NaCl plus 100 mM KCl (A Left, buffer Z), 100 mM KCl (A Right, mod buffer Z), or 150 mM KCl (B, buffer B). (A) In the absence of competitor RNA, binding of BC1 RNA to FMRP was observed under conditions of moderate but not high salt concentrations. (B) Binding of BC1 RNA, but of N19 RNA, was abolished in the presence of 100 ng/μl tRNA. Arrowheads indicate FMRP–RNA complexes. In addition, the positions of BC1 RNA and N19 RNA in the absence of FMRP are indicated. The asterisk indicates a minor species of differentially folded BC1 RNA formed under conditions of high salt concentrations. In general, whereas in vitro transcribed BC1 RNA resolves as a single band in denaturing gels, several bands reflecting different conformers are visualized in native gels, depending on salt concentrations. (C) Competition experiments for FMRP binding to BC1 RNA and N19 RNA were performed by using filter binding assays. Each data point represents the mean of three experimental values. The key lists RNAs in the format labeled/competitor. Error bars represent SEM. The data in this figure are from the E.W.K. laboratory.
Fig. 2.
Fig. 2.
BC1 RNA does not specifically interact with FMRP target mRNAs. Total RNA isolated from WT C57BL/6 mouse brains and from BC1−/− KO mouse brains was incubated with biotinylated BC1 RNA or an oligonucleotide complementary to the 3′ BC1 RNA region (bio-unique). The experimental protocol of Zalfa et al. (17) was followed throughout. RNA was extracted and RT-PCR was performed by using primers specific for the respective RNA species indicated. Lanes were loaded as follows: lane 1, total RNA from KO BC1−/− mouse brain was annealed to biotinylated BC1 RNA; lane 2, total RNA from KO BC1−/− mouse brain was annealed to biotinylated bio-unique oligonucleotide; lane 3, total RNA from KO BC1−/− mouse brain was mock-annealed to streptavidin magnetic beads; lane 4, total RNA from WT mouse brain was annealed to biotinylated BC1 RNA; lane 5, total RNA from WT mouse brain was annealed to biotinylated bio-unique oligonucleotide; lane 6, total RNA from WT mouse brain was mock-annealed to streptavidin magnetic beads; lane 7, total RNA from WT mouse brain was used as template for RT-PCR with specific oligonucleotides as a positive control for each experiment; lane 8, total RNA from KO BC1−/− mouse brain was used as template for RT-PCR with specific oligonucleotides as a positive control for each experiment. The data in this figure are from the J.B. laboratory.
Fig. 3.
Fig. 3.
U-rich FMRP target and nontarget mRNAs bind to poly(A) RNA. Mouse cortex total RNA (1 μg) was annealed to 20 μl of poly(A) RNA agarose resin, and bound RNA was converted to cDNA and amplified (see Materials and Methods). Results are shown in lane 3. Lane 4 shows controls performed in the absence of reverse transcriptase. Lanes 1 and 2 show amplifications of cDNA from total RNA (1 μg) after omission of the poly(A) binding step (reverse transcriptase omitted in lane 2). Lane 5 is an amplification of pET21A-FMRP plasmid DNA (10 ng). PD indicates primer–dimer bands, and asterisks indicate nonspecific bands. In the absence of poly(A) annealing (lanes 1 and 2), a minor band was in some cases apparent both in +RT and −RT lanes (e.g., dynamin A1 amplification). Bands in the −RT lane may indicate amplification from carryover of genomic DNA. However, such bands were not observed after poly(A) annealing. The data in this figure are from the R.B.D. laboratory.
Fig. 4.
Fig. 4.
FMRP associates with target mRNAs in vivo in the absence of BC1 RNA. Using monoclonal anti-FMRP antibody 7G1-1, coimmunoprecipitation was performed in brain extracts from WT and BC1−/− (KO) animals. (A) RNA was extracted and assayed for CaMKIIα, Arc, MAP1B, and Fmr1 mRNAs (i.e., FMRP target mRNAs) and for GAPDH mRNA (not an FMRP target). All examined FMRP target mRNAs were indistinguishably identified in WT and KO brains. Expected PCR product sizes were as follows: CaMKIIα, 354 bp; Arc, 86 bp; MAP1B, 119 bp; Fmr1, 134 bp; GAPDH, 233 bp. (B) Paired Student's t tests revealed no significant difference in FMRP target mRNA levels between WT and KO animals (P > 0.8 for each target mRNA; n ≥ 4 in all cases). For each group, the relative WT level was normalized to 1. The data in this figure are from the H.T. laboratory.
Fig. 5.
Fig. 5.
BC1 RNA does not associate with FMRP in vivo. (A) RT-PCR was performed with primers specific for BC1 RNA. Template RNA was extracted from input material obtained with WT (lane 1) and fmr1 KO (lane 2) brains, from irrelevant IgG-IP with WT brains (lane 3), from FMRP-IP (F-IP) with WT (lane 4) and fmr1 KO (lane 5) brains, and from PABP-IP (P-IP) with WT brains (lane 6). The BC1 PCR product (arrowhead) is detected in both input lanes and in the P-IP lane, but not in F-IP or IgG-IP lanes. PCR products marked by PD are primer dimers. The data in A are from the S.K. laboratory. (B) Northern hybridization was performed to probe for BC1 RNA in F-IP RNA and P-IP RNA. Whereas a strong signal was observed in the P-IP lane, no signal was detected in the F-IP lane (even after overexposure). In vitro transcribed BC1 RNA (10 ng) was used for reference (BC1 Ctr).The data in B are from the H.T. laboratory.
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Comment in

  • On BC1 RNA and the fragile X mental retardation protein.
    Bagni C. Bagni C. Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):E19. doi: 10.1073/pnas.0801034105. Epub 2008 Apr 15. Proc Natl Acad Sci U S A. 2008. PMID: 18417446 Free PMC article. No abstract available.
  • Reply to Bagni: On BC1 RNA and the fragile X mental retardation protein.
    Iacoangeli A, Rozhdestvensky TS, Dolzhanskaya N, Tournier B, Schütt J, Brosius J, Denman RB, Khandjian EW, Kindler S, Tiedge H. Iacoangeli A, et al. Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):E29. doi: 10.1073/pnas.0803737105. Epub 2008 May 29. Proc Natl Acad Sci U S A. 2008. PMID: 18511554 Free PMC article. No abstract available.

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