Anna Iacoangeli

Dendritic BC1 RNA in translational control mechanisms

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3′ domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3′ BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.

Poly(A)-binding protein is associated with neuronal BC1 and BC200 ribonucleoprotein particles.

BC1 RNA and BC200 RNA are two non-homologous, small non-messenger RNAs (snmRNAs) that were generated, evolutionarily, quite recently by retroposition. This process endowed the RNA polymerase III transcripts with central adenosine-rich regions. Both RNAs are expressed almost exclusively in neurons, where they are transported into dendritic processes as ribonucleoprotein particles (RNPs). Here, we demonstrate with a variety of experimental approaches that poly(A)-binding protein (PABP1), a regulator of translation initiation, binds to both RNAs in vitro and in vivo. We identified the association of PABP with BC200 RNA in a tri-hybrid screen and confirmed this binding in electrophoretic mobility-shift assays and via anti-PABP immunoprecipitation of BC1 and BC200 RNAs from crude extracts, immunodepleted extracts, partially purified RNPs and cells transfected with naked RNA. Furthermore, PABP immunoreactivity was localized to neuronal dendrites. Competition experiments using variants of BC1 and BC200 RNAs demonstrated that the central adenosine-rich region of both RNAs mediates binding to PABP. These findings lend support to the hypothesis that the BC1 and BC200 RNPs are involved in protein translation in neuronal dendrites.

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.

Spatial codes in dendritic BC1 RNA

BC1 RNA is a dendritic untranslated RNA that has been implicated in local translational control mechanisms in neurons. Prerequisite for a functional role of the RNA in synaptodendritic domains is its targeted delivery along the dendritic extent. We report here that the targeting-competent 5′ BC1 domain carries two dendritic targeting codes. One code, specifying somatic export, is located in the medial-basal region of the 5′ BC1 stem-loop structure. It is defined by an export-determinant stem-bulge motif. The second code, specifying long-range dendritic delivery, is located in the apical part of the 5′ stem-loop domain. This element features a GA kink-turn (KT) motif that is indispensable for distal targeting. It specifically interacts with heterogeneous nuclear ribonucleoprotein A2, a trans-acting targeting factor that has previously been implicated in the transport of MBP mRNA in oligodendrocytes and neurons. Our work suggests that a BC1 KT motif encodes distal targeting via the A2 pathway and that architectural RNA elements, such as KT motifs, may function as spatial codes in neural cells.

Translational control at the synapse: role of RNA regulators

Translational control of gene expression is instrumental in the regulation of eukaryotic cellular form and function. Neurons in particular rely on this form of control because their numerous synaptic connections need to be independently modulated in an input-specific manner. Brain cytoplasmic (BC) RNAs implement translational control at neuronal synapses. BC RNAs regulate protein synthesis by interacting with eIF4 translation initiation factors. Recent evidence suggests that such regulation is required to control synaptic strength, and that dysregulation of local protein synthesis precipitates neuronal hyperexcitability and a propensity for epileptogenic responses. A similar phenotype results from lack of fragile X mental retardation protein (FMRP), indicating that BC RNAs and FMRP use overlapping and convergent modes of action in neuronal translational regulation.

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).

Biased Immunoglobulin Light Chain Gene Usage in the Shark

This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ∼500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ∼39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing.

Regulatory BC1 RNA in cognitive control

Dendritic regulatory BC1 RNA is a non-protein-coding (npc) RNA that operates in the translational control of gene expression. The absence of BC1 RNA in BC1 knockout (KO) animals causes translational dysregulation that entails neuronal phenotypic alterations including prolonged epileptiform discharges, audiogenic seizure activity in vivo, and excessive cortical oscillations in the γ frequency band. Here we asked whether BC1 RNA control is also required for higher brain functions such as learning, memory, or cognition. To address this question, we used odor/object attentional set shifting tasks in which prefrontal cortical performance was assessed in a series of discrimination and conflict learning sessions. Results obtained in these behavioral trials indicate that BC1 KO animals were significantly impaired in their cognitive flexibility. When faced with conflicting information sources, BC1 KO animals committed regressive errors as they were compromised in their ability to disengage from recently acquired memories even though recall of such memories was in conflict with new situational context. The observed cognitive deficits are reminiscent of those previously described in subtypes of human autism spectrum disorders.

Evidence for Ig Light Chain Isotype Exclusion in Shark B Lymphocytes Suggests Ordered Mechanisms

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, κ, λ, σ, and σ-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)κ–expressing cells, isolated with mAb LK14 that recognizes Cκ, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM+ cells contained largely nonproductive κ and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional λ mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgκ+ splenocytes. Whereas λ somatic mutants from the depleted sample displayed evidence of positive selection, the λ genes in sIgκ+ cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: σ-2 followed by κ, then σ and λ.

BC RNA Mislocalization in the Fragile X Premutation

Abstract Fragile X premutation disorder is caused by CGG triplet repeat expansions in the 5′ untranslated region of FMR1 mRNA. The question of how expanded CGG repeats cause disease is a subject of continuing debate. Our work indicates that CGG-repeat structures compete with regulatory BC1 RNA for access to RNA transport factor hnRNP A2. As a result, BC1 RNA is mislocalized in vivo, as its synapto-dendritic presence is severely diminished in brains of CGG-repeat knock-in animals (a premutation mouse model). Lack of BC1 RNA is known to cause seizure activity and cognitive dysfunction. Our working hypothesis thus predicted that absence, or significantly reduced presence, of BC1 RNA in synapto-dendritic domains of premutation animal neurons would engender cognate phenotypic alterations. Testing this prediction, we established epileptogenic susceptibility and cognitive impairments as major phenotypic abnormalities of CGG premutation mice. In CA3 hippocampal neurons of such animals, synaptic release of glutamate elicits neuronal hyperexcitability in the form of group I metabotropic glutamate receptor–dependent prolonged epileptiform discharges. CGG-repeat knock-in animals are susceptible to sound-induced seizures and are cognitively impaired as revealed in the Attentional Set Shift Task. These phenotypic disturbances occur in young-adult premutation animals, indicating that a neurodevelopmental deficit is an early-initial manifestation of the disorder. The data are consistent with the notion that RNA mislocalization can contribute to pathogenesis.