BC1 RNA Research Articles

Application of the BC1 RNA gene promoter for short hairpin RNA expression in cultured neuronal cells

BC1 RNA is a neuronal cell-specific non-messenger RNA transcribed by RNA polymerase III (Pol III). We previously reported that the transcription of BC1 RNA is controlled both by intragenic promoters for Pol III and by a 5′-flanking region containing several unique cis-elements that are possible members of the Pol II transcription system. In this study, we chose β-secretase (BACE1) as a target and applied the promoter to produce a short hairpin RNA (shRNA) for RNA interference (RNAi) in cultured neuronal cells. A plasmid vector in which the promoter was linked to a target sequence functioned in rodent NG108-15 cells and suppressed BACE1 protein expression, but did not function in non-neuronal NIH3T3 cells. It was also effective in rat primary hippocampal neurons. We further showed that the promoter can be active in human neuroblastoma SH-SY5Y cells and reduced expression of targeted protein, although the BC1 RNA gene is a rodent-specific gene. The use of this vector-based system for shRNA expression may be an important component of future development of neuronal cell-selective RNAi in both transgenic and therapeutic applications.

Dendritic Transport and Localization of Protein Kinase Mζ mRNA: IMPLICATIONS FOR MOLECULAR MEMORY CONSOLIDATION

Protein kinase Mzeta (PKMzeta) is an atypical protein kinase C isoform that has been implicated in the protein synthesis-dependent maintenance of long term potentiation and memory storage in the brain. Synapse-associated kinases are uniquely positioned to promote enduring consolidation of structural and functional modifications at the synapse, provided that kinase mRNA is available on site for local input-specific translation. We now report that the mRNA encoding PKMzeta is rapidly transported and specifically localized to synaptodendritic neuronal domains. Transport of PKMzeta mRNA is specified by two cis-acting dendritic targeting elements (Mzeta DTEs). Mzeta DTE1, located at the interface of the 5'-untranslated region and the open reading frame, directs somato-dendritic export of the mRNA. Mzeta DTE2, in contrast, is located in the 3'-untranslated region and is required for delivery of the mRNA to distal dendritic segments. Colocalization with translational repressor BC1 RNA in hippocampal dendrites suggests that PKMzeta mRNA may be subject to translational control in local domains. Dendritic localization of PKMzeta mRNA provides a molecular basis for the functional integration of synaptic signal transduction and translational control pathways.

Dendritic BC1 RNA: modulation by kindling-induced afterdischarges

Local protein synthesis in dendrites is thought to provide a mechanism for long-lasting modifications of synapses in response to physiological activity and behavioral experience. New synthesis of dendritic proteins may be triggered by various paradigms, including induction of epileptiform activity. Prerequisite for such modulated synthesis is a mechanism that limits translation of synaptodendritic mRNAs to times of demand. Recently identified as a translational repressor that is localized to dendrites, small untranslated BC1 RNA has been implicated in the regulation of postsynaptic protein synthesis. Here we show that translational repressor BC1 RNA is itself undergoing modulation as a result of neuronal stimulation. Induction of hippocampal epileptiform activity resulted in a significant decrease of BC1 RNA in the CA3 region over several hours after excitation. The observed decrease was cell-wide, thus indicating reduced expression rather than intracellular redistribution. We suggest that a downregulation of the translational repressor BC1 RNA serves to modulate postsynaptic protein complements in response to the induction of epileptiform activity. Such increased protein synthesis in dendrites may be required for the consolidation of enduring epileptogenic mechanisms.

Role of a neuronal small non-messenger RNA: behavioural alterations in BC1 RNA-deleted mice

BC1 RNA is a small non-messenger RNA common in dendritic microdomains of neurons in rodents. In order to investigate its possible role in learning and behaviour, we compared controls and knockout mice from three independent founder lines established from separate embryonic stem cells. Mutant mice were healthy with normal brain morphology and appeared to have no neurological deficits. A series of tests for exploration and spatial memory was carried out in three different laboratories. The tests were chosen as to ensure that different aspects of spatial memory and exploration could be separated and that possible effects of confounding variables could be minimised. Exploration was studied in a barrier test, in an open-field test, and in an elevated plus-maze test. Spatial memory was investigated in a Barnes maze and in a Morris water maze (memory for a single location), in a multiple T-maze and in a complex alley maze (route learning), and in a radial maze (working memory). In addition to these laboratory tasks, exploratory behaviour and spatial memory were assessed under semi-naturalistic conditions in a large outdoor pen. The combined results indicate that BC1 RNA-deficient animals show behavioural changes best interpreted in terms of reduced exploration and increased anxiety. In contrast, spatial memory was not affected. In the outdoor pen, the survival rates of BC1-depleted mice were lower than in controls. Thus, we conclude that the neuron-specific non-messenger BC1 RNA contributes to the aptive modulation of behaviour.

Neuronal Untranslated BC1 RNA: Targeted Gene Elimination in Mice

Despite the potentially important roles of untranslated RNAs in cellular form or function, genes encoding such RNAs have until now received surprisingly little attention. One such gene encodes BC1 RNA, a small non-mRNA that is delivered to dendritic microdomains in neurons. We have now eliminated the BC1 RNA gene in mice. Three independent founder lines were established from separate embryonic stem cells. The mutant mice appeared to be healthy and showed no anatomical or neurological abnormalities. The gross brain morphology was unaltered in such mice, as were the subcellular distributions of two prototypical dendritic mRNAs (encoding MAP2 and CaMKIIalpha). Due to the relatively recent evolutionary origin of the gene, we expected molecular and behavioral consequences to be subtle. Behavioral analyses, to be reported separately, indicate that the lack of BC1 RNA appears to reduce exploratory activity.

The Fragile X Syndrome Protein FMRP Associates with BC1 RNA and Regulates the Translation of Specific mRNAs at Synapses

The Fragile X syndrome, which results from the absence of functional FMRP protein, is the most common heritable form of mental retardation. Here, we show that FMRP acts as a translational repressor of specific mRNAs at synapses. Interestingly, FMRP associates not only with these target mRNAs, but also with the dendritic, non-translatable RNA BC1. Blocking of BC1 inhibits the interaction of FMRP with its target mRNAs. Furthermore, BC1 binds directly to FMRP and can also associate, in the absence of any protein, with the mRNAs regulated by FMRP. This suggests a mechanism where BC1 could determine the specificity of FMRP function by linking the regulated mRNAs and FMRP. Thus, when FMRP is not present, loss of translational repression of specific mRNAs at synapses could result in synaptic dysfunction phenotype of Fragile X patients.

Positive and negative regulators for neuronal BC1 RNA transcription by RNA polymerase III are possible members of the RNA polymerase II transcription system

Neuronal cell-specific BC1 RNA is a unique RNA polymerase III (Pol III) transcript. The transcription is controlled by an activator E2 site and by BCRE, a repressor element, in response to neuronal activity. BC1 RNA is localized to dendritic domains as ribonucleoprotein particles, and it has been suggested to play a functional role in translational regulation of dendritic mRNAs. In the present study, using a luciferase assay in NG108-15 cells, we found that the positive and negative regulators for BC1 RNA transcription can also function in the Pol II transcription system. Our results suggest that the neuronal activity-dependent expression of BC1 RNA by Pol III and a subset of neuronal mRNAs by Pol II may be simultaneously controlled by the E2 site and BCRE, as well as their binding proteins.

Dendritic BC1 RNA: Functional Role in Regulation of Translation Initiation

In neurons, local protein synthesis in synaptodendritic microdomains has been implicated in the growth and plasticity of synapses. Prerequisites for local translation are the targeted transport of RNAs to distal sites of synthesis in dendrites and translational control mechanisms to limit synthesis to times of demand. Here we identify dendritic BC1 RNA as a specific repressor of translation. Experimental use of internal ribosome entry mechanisms and sucrose density gradient centrifugation showed that BC1-mediated repression targets translation at the level of initiation. Specifically, BC1 RNA inhibited formation of the 48S preinitiation complex, i.e., recruitment of the small ribosomal subunit to the messenger RNA (mRNA). However, 48S complex formation that is independent of the eukaryotic initiation factor 4 (eIF4) family of initiation factors was found to be refractory to inhibition by BC1 RNA, a result that implicates at least one of these factors in the BC1 repression pathway. Biochemical experiments indicated a specific interaction of BC1 RNA with eIF4A, an RNA unwinding factor, and with poly(A)-binding protein. Both proteins were found enriched in synaptodendritic microdomains. Significantly, BC1-mediated repression was shown to be effective not only in cap-dependent translation initiation but also in eIF4-dependent internal initiation. The results suggest a functional role of BC1 RNA as a mediator of translational control in local protein synthesis in nerve cells.

Binding of p190RhoGEF to a Destabilizing Element on the Light Neurofilament mRNA Is Competed by BC1 RNA

The enhancement of RNA-mediated motor neuron degeneration in transgenic mice by mutating a major mRNA instability determinant in a light neurofilament (NF-L) transgene implicates cognate RNA binding factors in the pathogenesis of motor neuron degeneration. p190RhoGEF is a neuron-enriched guanine exchange factor (GEF) that binds to the NF-L-destabilizing element, to c-Jun N-terminal kinase-interactive protein-1 (JIP-1), and to 14-3-3 and may link neurofilament expression to pathways affecting neuronal homeostasis. This study was undertaken to identify additional RNA species that bind p190RhoGEF and could affect interactions of the exchange factor with NF-L transcripts. The C-terminal domain of p190RhoGEF, containing the RNA-binding site, was expressed as a glutathione S-transferase fusion protein and was used as an affinity probe to isolate interactive RNAs in rat brain extracts. As expected, NF-L mRNA was identified as an RNA specie eluted from the affinity column. In addition, BC1 RNA was also found enriched in the bound RNA fraction. BC1 is a 152-nucleotide RNA that is highly expressed but untranslated in differentiated neurons. We show that BC1 and NF-L mRNA bind to a similar site in the C-terminal domain of p190RhoGEF, and their bindings to p190RhoGEF are readily cross-competed. Moreover, we identify a novel binding site in BC1 to account for its interaction with p190RhoGEF. The findings suggest a novel role of BC1 in differentiated neurons involving RNA-protein interactions of p190RhoGEF.

Identification of mRNA/Protein (mRNP) Complexes Containing Purα, mStaufen, Fragile X Protein, and Myosin Va and their Association with Rough Endoplasmic Reticulum Equipped with a Kinesin Motor

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.

Shared Protein Components of SINE RNPs

The heterogeneous, short RNAs produced from the high, copy, short mobile elements (SINEs) interact with proteins to form RNA–protein (RNP) complexes. In particular, the BC1 RNA, which is transcribed to high levels specifically in brain and testis from one locus of the ID SINE family, exists as a discrete RNP complex. We expressed a series of altered BC1, and other SINE-related RNAs, in several cell lines and tested for the mobility of the resulting RNP complexes in a native PAGE assay to determine which portions of these SINE RNAs contribute to protein binding. When different SINE RNAs were substituted for the BC1 ID sequence, the resulting RNPs exhibited the same mobility as BC1. This indicates that the protein(s) binding to the ID portion of BC1 is not sequence specific and may be more dependent upon the secondary structure of the RNA. It also suggests that all SINE RNAs may bind a similar set of cellular proteins. Deletion of the A-rich region of BC1 RNA has a marked effect on the mobility of the RNP. Rodent cell lines exhibit a slightly different mobility for this shifted complex when compared to human cell lines, reflecting evolutionary differences in one or more of the protein components. On the basis of mobility change observed in RNP complexes when the A-rich region is removed, we decided to examine poly(A) binding protein (PABP) as a candidate member of the RNP. An antibody against the C terminus of PABP is able to immunoprecipitate BC1 RNA, confirming PABP's presence in the BC1 RNP. Given the ubiquitous role of poly(A) regions in the retrotransposition process, these data suggest that PABP may contribute to the SINE retrotransposition process.

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.

Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

Transport of Neuronal BC1 RNA in Mauthner Axons

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons <i>in vivo</i>. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 μm/sec. Whereas a BC1 5′ segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3′ segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

A small RNA in testis and brain: implications for male germ cell development.

BC1 RNA, a small non-coding RNA polymerase III transcript, is selectively targeted to dendritic domains of a subset of neurons in the rodent nervous system. It has been implicated in the regulation of local protein synthesis in postsynaptic microdomains. The gene encoding BC1 RNA has been suggested to be a master gene for repetitive ID elements that are found interspersed throughout rodent genomes. A prerequisite for the generation of repetitive elements through retroposition and subsequent transmission in the germline is expression of the master gene RNA in germ cells. To test this hypothesis, we have investigated expression of BC1 RNA in murine male germ cells. We report that BC1 RNA is expressed at substantial levels in a subset of male germ cells. Results from cell fractionation experiments, developmental analysis, and northern and in situ hybridization showed that the RNA was expressed in pre-meiotic spermatogonia, with particularly high amounts in syncytial ensembles of cells that are primed for synchronous spermatogenic differentiation. BC1 RNA continued to be expressed in spermatocytes, but expression levels decreased during further spermatogenic development, and low or negligible amounts of BC1 RNA were identified in round and elongating spermatids. The combined data indicate that BC1 RNA operates in groups of interconnected germ cells, including spermatogonia, where it may function in the mediation of translational control. At the same time, the identification of BC1 RNA in germ cells provides essential support for the hypothesis that repetitive ID elements in rodent genomes arose from the BC1 RNA gene through retroposition.

Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

Neuronal BC1 RNA: Co-expression with growth-associated protein-43 messenger RNA

Brain-specific cytoplasmic RNA 1 (BC1-RNA), a non-coding RNA polymerase III transcript, is a neuronal RNA that is specifically targeted to dendritic domains. It is co-localized with components of the dendritic protein synthetic machinery, and it has been suggested to operate in the regulation of local translation-related processes in postsynaptic microdomains, thus subserving long-term synaptic plasticity in neurons. To probe the relevance of BC1 expression in neuronal plasticity, we have analyzed the expression pattern of BC1 RNA in the rat nervous system. We found that BC1 RNA is expressed by a specific subset of neurons (but not by non-neuronal cells) in the central and peripheral nervous system of the adult rat. The BC1 labeling pattern indicates that the subcellular location of the RNA is typically postsynaptic which, depending on cell type, manifests itself in a predominantly somatic, somatodendritic, or dendritic location. Our results further show that BC1-expressing neurons typically co-express the messenger RNA for growth-associated protein-43 (GAP-43). Such co-expression was observed in diverse brain areas, including the olfactory bulb, neocortex, and hippocampus, among others. While BC1 RNA was in many neuronal cell types detectable in distal dendritic domains, GAP-43 messenger RNA was typically more restricted to neuronal perikarya. In the mature nervous system, expression of GAP-43 has been described as an intrinsic determinant of predominantly presynaptic plasticity, while BC1 RNA has been implicated in postsynaptic plasticity. Co-expression of both RNAs, as reported here, thus identifies a distinct subset of neurons in the rat nervous system that exhibits both types of plasticity.

Pur alpha protein implicated in dendritic RNA transport interacts with ribosomes in neuronal cytoplasm.

We have previously reported that pur alpha, known to be a regulator of DNA replication and transcription, links neural BC1 RNA to microtubules via dendrite-targeting RNA motifs. Here we demonstrate the subcellular localization of pur proteins within the brain. Pur proteins were detected in neurons but not in glia. Immunohistochemical staining was prominent in perikarya and proximal dendrites and also extended into primary dendritic processes, but no significant signals were detected in the distal regions of dendrite. When homogenates of mouse brain were fractionated, pur alpha was most concentrated in the microsomal pellet. Consistently, pur alpha co-fractionated with free polysomes as well as with membrane-bound polysomes and the association with polysomes was mediated by binding ribosomal subunits. Levels of ribosomes with pur alpha progressively increased during postnatal development of the brain.

The single-stranded DNA- and RNA-binding proteins pur alpha and pur beta link BC1 RNA to microtubules through binding to the dendrite-targeting RNA motifs.

Neural BC1 RNA is distributed in neuronal dendrites as RNA-protein complexes (BC1 RNPs) containing Translin. In this study, we demonstrated that the single-stranded DNA- and RNA-binding protein pur alpha and its isoform, pur beta, which have been implicated in control of DNA replication and transcription, linked BC1 RNA to microtubules (MTs). The binding site was within the 5' proximal region of BC1 RNA containing putative dendrite-targeting RNA motifs rich in G and U residues, suggesting that in the cytoplasm of neurons, these nuclear factors are involved in the BC1 RNA transport along dendritic MTs. The pur proteins were not components of BC1 RNP but appeared to associate with MTs in brain cells. Therefore, it is suggested that they may transiently interact with the RNP during transport. In this respect, the interaction of pur proteins with BC1 RNA could be regulated by the Translin present within the RNP, because the binding mode of these two classes of proteins (pur proteins and Translin) to the dendrite-targeting RNA motifs was mutually exclusive. As the motifs are well conserved in microtubule-associated protein 2a/b mRNA as well, the pur proteins may also play a role(s) in the dendritic transport of a subset of mRNAs.

Neural BC1 RNA Associates with Pur α, a Single-Stranded DNA and RNA Binding Protein, Which Is Involved in the Transcription of the BC1 RNA Gene

BC1 RNA is preferentially expressed in neural cells by RNA polymerase III (Pol III) and forms ribonucleoprotein particles (RNP) in the somatodendritic domain of neurons. Our previous studies have suggested that, in the nucleus, BC1 RNA forms an RNP containing a nuclear protein(s) that participates in the transcription of the BC1 RNA gene. In this study, we have shown that newly synthesized BC1 RNA in purified brain nuclear extracts is immunoprecipitated by an antibody against Pur α. Pur α is a protein that binds single-stranded DNA and RNA and is known to regulate transcription of Pol II system. Although BC1 RNA is transcribed by Pol III, the BC1 RNA gene has two putative Pur α binding sites, which Pur α specifically recognizes. Point mutations within these sites reduced transcriptional activity in vitro. Furthermore, transcription was inhibited by depletion of Pur α from the nuclear extracts, either by the coexistence of its binding region of BC1 RNA or by the antibody that was able to precipitate the nuclear BC1 RNP. These observations suggest that BC1 RNA associates with Pur α which is involved in the transcription of the BC1 RNA gene.