Natalia Dolzhanskaya

On BC1 RNA and the fragile X mental retardation protein

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 FMRPs 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.

Methylation regulates the intracellular protein-protein and protein-RNA interactions of FMRP.

FMRP, the fragile X mental retardation protein, is an RNA-binding protein that interacts with ∼4% of fetal brain mRNA. We have recently shown that a methyltransferase (MT) co-translationally methylates FMRP in vitro and that methylation modulates the ability of FMRP to bind mRNA. Here, we recapitulate these in vitro data in vivo, demonstrating that methylation of FMRP affects its ability to bind to FXR1P and regulate the translation of FMRP target mRNAs. Additionally, using double-label fluorescence confocal microscopy, we identified a subpopulation of FMRP-containing small cytoplasmic granules that are distinguishable from larger stress granules. Using the oxidative-stress induced accumulation of abortive pre-initiation complexes as a measure of the association of FMRP with translational components, we have demonstrated that FMRP associates with ribosomes during initiation and, more importantly, that methylation regulates this process by influencing the ratio of FMRP-homodimer-containing mRNPs to FMRP-FXR1P-heterodimer-containing mRNPs. These data suggest a vital role for methylation in normal FMRP functioning.

The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system ☆

We recently identified several ESTs that bind to the fragile X mental retardation protein (FMRP) in vitro. To determine whether they interacted in vivo we performed three-hybrid screens in a Saccharomyces cerevisiae histidine auxotroph. We demonstrate that two of the ESTs support growth on histidine and transduce beta-galactosidase activity when co-expressed with FMRP under selective growth conditions. In contrast, the iron response element (IRE) RNA does not. Likewise, the ESTs do not support growth or transduce beta-galactosidase activity when co-expressed with the iron response element binding protein (IRP). Each EST is relatively small and has 40% identity with a sequence in FMR1 mRNA harboring FMRP binding determinants. Interestingly, while neither the ESTs contain a G-quartet structural motif they do contain U-rich sequences that are found in mRNA with demonstrated in vitro binding and in vivo association with FMRP. This indicates that U-rich elements comprise another motif recognized by FMRP.

Tissue and developmental regulation of fragile X mental retardation 1 exon 12 and 15 isoforms

The pre-mRNA of the fragile X mental retardation 1 gene (FMR1) is subject to exon skipping and alternative splice site selection, which can generate up to 12 isoforms. The expression and function of these variants in vivo has not yet been fully explored. In the present study, we investigated the distribution of Fmr1 exon 12 and exon 15 isoforms. Exon 12 encodes an extension of KH(2) domain, one of the RNA binding domains in the FMR1 gene product (FMRP) and we show that exon 12 variant proteins differentially interact with kissing complex RNA. Alternative splicing at exon 15 produces FMRPs differing in RNA binding ability and each is distinguished by unique post-translational modifications. Using semiquantitative RT-PCR and Northern blotting, we found that particular Fmr1 exon 12 and exon 15 isoforms change during neuronal differentiation. Interestingly, Fmr1 exon 12 variants display tissue-specific and developmental differences, while exon 15-containing transcripts vary less. Altogether, the spatio-temporal plasticity of FMR1 mRNA is consistent with complex RNA processing that is mis-regulated in fragile X syndrome.

Regulating a translational regulator: mechanisms cells use to control the activity of the fragile X mental retardation protein

Fragile X syndrome results from the loss of a normal cellular protein, FMRP. FMRP is an RNA binding protein, and it is likely that altering the way FMRP’s messenger RNA (mRNA) targets are processed results in the clinical features associated with the disease. Using complementary DNA microarray screening, a number of brain-derived mRNAs that interact directly with FMRP in vitro and associate with FMRP-containing mRNPs in vivo have been identified. These target messages encode RNA-binding proteins, transcription factors, neuronal receptors, cytoskeletal proteins, a few enzymes as well as several unknown proteins. For a subset of these mRNAs it has been shown that modulating FMRP levels in cultured cells correspondingly affects their expression. In addition, several modes by which cells modulate FMRP activity have been described; these include posttranscriptional processing and posttranslational modification. Here, the most recent results concerning the biochemical activities of FMRP and how they are affected by various modifications are reviewed. The data lead to a model signaling mechanism by which FMRP normally regulates the expression of its target mRNAs.

Prosegment of tripeptidyl peptidase I is a potent, slow-binding inhibitor of its cognate enzyme.

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.

Oxidative stress reveals heterogeneity of FMRP granules in PC12 cell neurites

PC12 cells are a well-known model of parasympathetic neurons. They have also been used to study the dynamics of heterologously expressed fragile X mental retardation (FMRP) granule trafficking down neurites. Here, we demonstrate that undifferentiated and differentiated PC12 cells harbor endogenous FMRP-containing granules. These granules are not stress granules because they do not associate with an authentic stress granule marker protein T-cell internal antigen 1 (TIA-1). Treatment with sodium arsenite induces stress granule formation in undifferentiated and differentiated PC12 cells. In NGF-treated cells, FMRP-containing stress granules are observed in the soma, neurites and growth cones by co-immunostaining with anti-TIA-1 antibody. These data demonstrate that all three microdomains respond similarly to oxidative stress. Nevertheless, we find significantly less co-localization of FMRP and TIA-1 and FMRP and its homologs in the neurites of differentiated PC12 cells treated with sodium arsenite than in the soma or growth cones. The heterogeneity of these granules suggests that FMRP has multiple roles in neurites.

Reply to Bagni: On BC1 RNA and the fragile X mental retardation protein

In Iacoangeli et al. (1), five independent groups report that results published by Zalfa et al. (2) are not reproducible. Bagni now suggests (3) that different reagents, antibodies, or procedures might explain this lack of reproducibility. Iacoangeli et al. replicated the experimental conditions reported by Zalfa et al. whenever possible, as indicated. In several cases, however, reagents used by Zalfa et al. were not available. For instance, Zalfa et al. generated antibody rAM1 and used it to probe BC1–FMRP interactions in supershift and other assays. The antibody did not produce a supershift in brain extracts—as one would have expected if BC1 RNA did in fact bind to FMRP in vivo—but abolished the regular mobility shift (2). Despite repeated requests by several of the undersigned, antibody rAM1 was not provided. We urge the Bagni group (2) to make antibody rAM1 available for independent examination. In contrast, Iacoangeli et al. used two established anti-FMRP antibodies that have been independently validated and are publicly available. Bagni suggests that two other groups (4, 5) have published data in support of her claims. Both articles were quoted and discussed by Iacoangeli et al. (1). One of the undersigned (E.W.K.) coauthored one of these articles (4) and has confirmed that, although BC1 RNA does bind to FMRP in vitro, this binding is entirely nonspecific because it is completely abrogated by competitor tRNA (1). Bagni does not mention that Gabus et al. (4) reported a Kd of FMRP for tRNA of 25 nM, the same as for BC1 RNA. The second article (5) reported a weak BC1 RT-PCR signal in FMRP cross-linked immunoprecipitates and a stronger signal in MAP2 cross-linked immunoprecipitates. However, considering that MAP2 is not a known RNA binding protein, mere copurification cannot be taken as evidence for physical association (1). Thus, there is no confirmation, independent of the Bagni group, of a specific physical link between FMRP and BC1 RNA, as posited by Zalfa et al. (2). Similarly, the Zalfa et al. claim that FMRP is not associated with polyribosomes in neurons could not be confirmed in subsequent work (6, 7).