BC1 RNA Research Articles

Identification of a negative regulatory DNA element for neuronal BC1 RNA expression by RNA polymerase III

BC1 RNA is a neuronal cell-specific RNA polymerase III (Pol III) transcript. The BC1 RNA gene has plural types of Pol III promoters, in addition to which an E-box sequence (E2 site) acts as a transcriptional activator, which is recognized by a brain-specific protein(s). Using an in vitro transcription system, we found that the upstream region of the BC1 RNA gene contained a sequence that interfered with the activity of the E-box element in a distance-independent manner. A tandem repeat within this sequence, which was weakly homologous with the neuron-restrictive silencer element (NRSE) found in the Pol II system, was recognized by a brain nuclear protein. Consistently, the transcriptional activity increased by deleting the tandem repeat sequence. We called this BC1 RNA-repressing element BCRE. The DNA-binding specificities of BCRE-binding protein differed from that of NRSE-binding protein (NRSF). A similar protein with an ability to bind to BCRE was also found in liver and kidney. Furthermore, the glutamate analog kainic acid increased the DNA-binding of both E2 site-binding protein and BCRE-binding protein, and then the levels of BC1 RNA also increased transiently. Our results suggested that both positive and negative regulatory elements contribute to neuronal BC1 RNA expression.

Upstream flanking sequences and transcription of SINEs

SINEs, short interspersed repeated DNA elements, undergo amplification through retroposition and subsequent integration into a new location in the genome. Each new SINE insertion will be located in a new chromosomal environment, with different flanking sequences. Modulation of transcription by different flanking sequences may play an important role in determining which SINE elements are preferentially active in a genome. We evaluated the ability of upstream flanking sequences to regulate the transcription of three different SINEs (Alu, B2 and ID) by constructing chimeric constructs with known 5′ flanking sequences of RNA polymerase III-transcribed genes. Upstream sequences from the 7SL RNA gene, U6 RNA gene, vault RNA gene, and BC1 gene increase transcription of Alu, B2 and BC1 in transient transfections of NIH3T3, HeLa, Neuro2a and C6 glioma cell lines. The 7SL sequence proved most efficient in increasing SINE transcription. The 7SL upstream fused to the BC1 RNA gene (an ID element) was used to create a transgenic mouse line. In contrast to the tissue-specific endogenous BC1 transcription, BC1 transgene transcripts were detected in all tissues tested. However, expression was much higher in those tissues that express the endogenous gene, demonstrating both transcriptional and post-transcriptional regulation. The BC1 RNA was detected in a similar ribonucleoprotein complex in the different tissues.

RNAs from all categories generate retrosequences that may be exapted as novel genes or regulatory elements.

While the significance of middle repetitive elements had been neglected for a long time, there are again tendencies to ascribe most members of a given middle repetitive sequence family a functional role--as if the discussion of SINE (short interspersed repetitive elements) function only can occupy extreme positions. In this article, I argue that differences between the various classes of retrosequences concern mainly their copy numbers. Consequently, the function of SINEs should be viewed as pragmatic such as, for example, mRNA-derived retrosequences, without underestimating the impact of retroposition for generation of novel protein coding genes or parts thereof (exon shuffling by retroposition) and in particular of SINEs (and retroelements) in modulating genes and their expression. Rapid genomic change by accumulating retrosequences may even facilitate speciation [McDonald, J.F., 1995. Transposable elements: possible catalysts of organismic evolution. Trends Ecol. Evol. 10, 123-126.] In addition to providing mobile regulatory elements, small RNA-derived retrosequences including SINEs can, in analogy to mRNA-derived retrosequences, also give rise to novel small RNA genes. Perhaps not representative for all SINE/master gene relationships, we gained significant knowledge by studying the small neuronal non-messenger RNAs, namely BC1 RNA in rodents and BC200 RNA in primates. BC1 is the first identified master gene generating a subclass of ID repetitive elements, and BC200 is the only known Alu element (monomeric) that was exapted as a novel small RNA encoding gene.

The Dendritic Translocation of Translin Protein in the Form of BC1 RNA Protein Particles in Developing Rat Hippocampal Neurons in Primary Culture

Neural BC1 RNA is distributed in neuronal dendrites as ribonucleoprotein particles (RNP). Our previous studies indicated the presence of Translin in BC1 RNPs, which is a translational repressor and links a subset of mRNAs to microtubules. In this study, we confirmed that Translin associates with BC1 RNP and we used immunocytochemical methods to examine the subcellular distribution of Translin in developing hippocampal cells in primary cultures. Translin was detected in both the nuclei and cytoplasm of neurons, whereas in glial cells it was localized in the nuclei. Consistent with the reported developmental time course of BC1 RNA expression and dendritic delivery the translocation of Translin to the neuronal dendrites appeared to correlate with neuronal development and differentiation events such as the onset of synaptogenesis in culture. These observations suggest that BC1 RNP or Translin itself may be relevant to the dendritic translation of mRNAs in response to transsynaptic activity.

Identification of human autoantigen La/SS-B as BC1/BC200 RNA-binding protein.

Rodent BC1 RNA and primate BC200 RNA are small cytoplasmic non-messenger RNAs that are phylogenetically unrelated. Nevertheless, the two RNAs exhibit a large degree of parallelism. In addition to some sequence similarities in their 3' domains, they are prevalently expressed in a similar subset of neurons and belong to a small group of transcripts with a somatodendritic location. Both RNAs are complexed with proteins as ribonucleoprotein particles (RNPs). Their similarities may even extend to analogous functional roles, for example, in the regulation of decentralized dendritic translation. To shed further light on the physiological role(s) of the BC1/BC200 RNPs, we began to analyze protein components that specifically bind to these RNAs. Ultraviolet-crosslinking experiments and affinity purification techniques revealed that the human autoantigen La/SS-B is associated with BC1/BC200 RNA in vitro and in vivo. As with other RNA polymerase III transcripts, La protein binds with high affinity to the 3' end of BC200 RNA. Our results suggest that an additional function of La may be control of dendritic translation by providing a link between the 5' Alu domain of BC200 RNP and the ribosome via the La protein dimer. The fact that La binds both BC1 and BC200 RNAs further supports the notion that the RNAs are functional analogs despite the fact that they arose from two separate retroposition events in two different mammalian lineages.

Activity-dependent regulation of dendritic BC1 RNA in hippocampal neurons in culture.

Several neuronal RNAs have been identified in dendrites, and it has been suggested that the dendritic location of these RNAs may be relevant to the spatiotemporal regulation of mosaic postsynaptic protein repertoires through transsynaptic activity. Such regulation would require that dendritic RNAs themselves, or at least some of them, be subject to physiological control. We have therefore examined the functional regulation of somatodendritic expression levels of dendritic BC1 RNA in hippocampal neurons in culture. BC1 RNA, an RNA polymerase III transcript that is a component of a ribonucleoprotein particle, became first detectable in somatodendritic domains of developing hippocampal neurons at times of initial synapse formation. BC1 RNA was identified only in such neurons that had established synapses on cell bodies and/or developing dendritic arbors. When synaptic contact formation was initiated later in low-density cultures, BC1 expression was coordinately delayed. Inhibition of neuronal activity in hippocampal neurons resulted in a substantial but reversible reduction of somatodendritic BC1 expression. We conclude that expression of BC1 RNA in somatic and dendritic domains of hippocampal neurons is regulated in development, and is dependent upon neuronal activity. These results establish (for the first time to our knowledge) that an RNA polymerase III transcript can be subject to control through physiological activity in nerve cells.

BC1 RNA Protein Particles in Mouse Brain Contain Two Y-,H-Element-Binding Proteins, Translin and a 37 kDa Protein

Neural BC1 RNA is distributed in dendrites in the form of ribonucleoprotein particles (RNP). Recently, Hanet al.reported that testis-brain RNA-binding protein (the mouse homologue of Translin), known to be a translational repressor, links a subset of mRNAs to microtubules (MTs) through binding to their y-,h-sequence elements. We found that similar elements are also present in BC1 RNA. Therefore, it is possible that they also act ascis-elements, together with their transacting proteins, and control the transport of BC1 RNA along dendritic MTs. In this study, we demonstrated that two y-,h-element-binding proteins copurified with BC1 RNP. One was identified as mouse Translin and the other was a 37 kDa protein. This macromolecular assembly may constitute a transport particle for BC1 RNA. It is also possible that BC1 RNP or Translin itself may play a regulatory role(s) in the translation of mRNAs within dendrites.

An E-Box Sequence Acts as a Transcriptional Activator for BC1 RNA Expression by RNA Polymerase III in the Brain

BC1 RNA is a small cytoplasmic RNA that is transcribed by RNA polymerase III (Pol III) in the rodent nervous system. In addition to essential intragenic promoter elements for Pol III, the BC1 RNA gene has five E-box sequences (CANNTG) in its 5′ flanking region. Deletion analysis using anin vitrotranscription system revealed that the region containing the E2 site (CAATTG) was necessary for effective transcription of BC1 RNA. A construct with point mutations within the E2 site showed reduced transcriptional activity. Furthermore, DNaseT I protection and gel retardation assays demonstrated that the E2 site was recognized specifically by a brain nuclear protein(s). These results suggest that the upstream E-box sequence and its binding protein may be involved in the regulation by Pol III of preferential BC1 RNA expression in the brain.

Gene delivery to central nervous system (CNS) by retrograde transport of recombinant replication-competent and aneurovirulent herpes simplex virus type 1, β strain

BC1 RNA is a neuronal cell-specific RNA polymerase III (Pol III) transcript. The BC1 RNA gene has plural types of Pol III promoters, in addition to which an E-box sequence (E2 site) acts as a transcriptional activator, which is recognized by a brain-specific protein(s). Using an in vitro transcription system, we found that the upstream region of the BC1 RNA gene contained a sequence that interfered with the activity of the E-box element in a distance-independent manner. A tandem repeat within this sequence, which was weakly homologous with the neuron-restrictive silencer element (NRSE) found in the Pol II system, was recognized by a brain nuclear protein. Consistently, the transcriptional activity increased by deleting the tandem repeat sequence. We called this BC1 RNA-repressing element BCRE. The DNA-binding specificities of BCRE-binding protein differed from that of NRSE-binding protein (NRSF). A similar protein with an ability to bind to BCRE was also found in liver and kidney. Furthermore, the glutamate analog kainic acid increased the DNA-binding of both E2 site-binding protein and BCRE-binding protein, and then the levels of BC1 RNA also increased transiently. Our results suggested that both positive and negative regulatory elements contribute to neuronal BC1 RNA expression.

Mutational Analysis Reveals That an Array of GCAAG/CTTGC Motifs between Sprit Promoter Sequences for RNA Polymerase III Is Essential for Neural BC1 RNA Transcription

BC1 RNA is expressed from an identifier (ID) sequence by RNA polymerase III (Pol III) and occurs in neural cells as a ribonucleoprotein particle (BC1 RNP). On the BC1 RNA gene, between the Pol III promoter A and B boxes, there is a region which contains short inverted repeats, including three GCAAG/CTTGC motifs. We found that a nuclear protein binds specifically to this region and, using anin vitrotranscription system, demonstrated that point mutations within these motifs markedly inhibit BC1 RNA transcription. These results suggest that the GCAAG/CTTGC motif region and its binding protein may play a role in the transcription of BC1 RNA. Moreover, we demonstrated that transcription is repressed by a concomitant molar excess of BC1 RNA and that the BC1 RNA transcribed by this system forms an RNP with nuclear protein(s), suggesting some interaction of BC1 RNA with transcription factor(s).

Localization of the Mouse Gene (Bc1) Encoding Neural BC1 RNA Near the Fibroblast Growth Factor 3 Locus (Fgf3) on Distal Chromosome 7

Localization of the Mouse Gene (Bc1) Encoding Neural BC1 RNA Near the Fibroblast Growth Factor 3 Locus (Fgf3) on Distal Chromosome 7

RNA Transport in Dendrites: Acis-Acting Targeting Element Is Contained within Neuronal BC1 RNA

In nerve cells, a select group of RNAs has been localized to dendritic domains. Here we have examined dendritic RNA transport in sympathetic neurons in primary culture, using a microinjection protocol with neuronal BC1 RNA and with BC1-derived sequence segments. After cytoplasmic microinjection, full-length BC1 RNA was selectively transported to dendrites; in contrast, control RNAs such as nuclear RNAs and random-sequence irrelevant RNAs remained restricted to cytoplasmic areas proximal to the injection sites. Chimeric RNAs were constructed that contained the full-length BC1 sequence inserted upstream or downstream of the coding regions of nondendritic mRNAs. After microinjection, such chimeric RNAs were specifically targeted to dendrites; microinjected corresponding nonchimeric mRNAs were not. Dendritic transport of BC1 RNA was rapid: the average dendritic delivery rate within the first hour after microinjection was 242 +/- 25 microm/hr. Whereas a 5'-BC1 segment of 62 nucleotides was transported to dendrites to extents and at levels similar to full-length BC1 RNA, a 3'-BC1 segment of 60 nucleotides did not exit injected somata to any significant degree. A cis-acting dendritic targeting element is thus contained in the 5' part of neuronal BC1 RNA. These results demonstrate that mechanisms exist in neurons for fast and specific transport of selected RNAs to dendrites.

BC1 RNA, the transcript from a master gene for ID element amplification, is able to prime its own reverse transcription.

ID elements are short interspersed elements (SINEs) found in high copy number in many rodent genomes. BC1 RNA, an ID-related transcript, is derived from the single copy BC1 RNA gene. The BC1 RNA gene has been shown to be a master gene for ID element amplification in rodent genomes. ID elements are dispersed through a process termed retroposition. The retroposition process involves a number of potential regulatory steps. These regulatory steps may include transcription in the appropriate tissue, transcript stability, priming of the RNA transcript for reverse transcription and integration. This study focuses on priming of the RNA transcript for reverse transcription. BC1 RNA gene transcripts are shown to be able to prime their own reverse transcription in an efficient intramolecular and site-specific fashion. This self-priming ability is a consequence of the secondary structure of the 3'-unique region. The observation that a gene actively amplified throughout rodent evolution makes a RNA capable of efficient self-primed reverse transcription strongly suggests that self-priming is at least one feature establishing the BC1 RNA gene as a master gene for amplification of ID elements.

Expression of neural BC1 RNA: induction in murine tumours.

BC1 RNA is a small cytoplasmic RNA polymerase III transcript that is expressed in the rodent nervous system. The RNA is selectively expressed in neurons where it is located in somatodendritic domains. BC1 RNA is not normally detectable in non-neuronal somatic cells; it is however expressed in germ cells and in cultured immortal cell lines of various non-neural origins. We therefore sought to establish whether the neuron-specific regulation of BC1 expression is altered in non-neural tumour cells. Oncogen and chemical carcinogen induced mouse tumours were analysed for the presence of BC1 RNA, using Northern transfer and in situ hybridisation. Here we report that BC1 RNA is selectively expressed in tumour cells, but not in corresponding normal tissues. These results indicate that neural-specific regulation of BC1 expression is lacking in murine tumour cells of non-neural origin.

Neural BC1 RNA in mouse skeletal muscle is a denervation-induced RNA whose expression is developmentally regulated

We detected neural BC1 RNA in mouse skeletal muscle. The level of BC1 RNA was high in the fetus, but it declined progressively to the adult level as development proceeded. These observations suggest that this RNA is involved in the prenatal development and differentiation of muscles. Although its developmental expression correlates with the fetal period of polyneuronal innervation, BC1 RNA does not seem to play a direct role(s) in synaptogenesis, since its expression was not restricted to the neuromuscular junction. We also demonstrated that the BC1 RNA level in adult muscle was elevated after denervation, suggesting that changes in the activity of muscles or neural factors caused by axotomy, or both may results in BC1 RNA upregulation.

Neural BC1 RNA in mouse skeletal muscle is a denervation-induced RNA whose expression is developmentally regulated

We detected neural BC1 RNA in mouse skeletal muscle. The level of BC1 RNA was high in the fetus, but it declined progressively to the adult level as development proceeded. These observations suggest that this RNA is involved in the prenatal development and differentiation of muscles. Although its developmental expression correlates with the fetal period of polyneuronal innervation, BC1 RNA does not seem to play a direct role(s) in synaptogenesis, since its expression was not restricted to the neuromuscular junction. We also demonstrated that the BC1 RNA level in adult muscle was elevated after denervation, suggesting that changes in the activity of muscles or neural factors caused by axotomy, or both may results in BC1 RNA upregulation.

Evolution, Expression, and Possible Function of a Master Gene for Amplification of an Interspersed Repeated DNA Family in Rodents

Publisher Summary BC1 RNA was originally identified as a small cytoplasmic RNA species, found primarily within the brain of rats that hybridized with a major—short interspersed repetitive element (SINE)— family of repetitive DNA sequences in the rat genome. A number of proposals have been made concerning the relationship of this single BC1 locus and other dispersed ID elements, both in terms of functional models and evolutionary relationships. Although no specific function has been demonstrated for either the BC1 RNA or ID elements, a number of lines of investigation suggest that the BC1 RNA gene plays some functional role, probably within neurons, throughout the rodent order. The BC1 RNA gene has also been shown to be a master gene for ID repeat amplification and evolution. The BC1 RNA gene is responsible for a large portion of the amplification of ID repeats via retroposition. There are two important aspects of the conservation of the BC1 RNA gene. The first is the conservation of transfer RNA-like features within the BC1 RNA gene. The second is the evidence that conservation provides regarding regions that may be under functional selection.

Transcription and processing of the rodent ID repeat family in germline and somatic cells.

ID elements comprise a rodent SINE (short interspersed DNA repetitive element) family that has amplified by retroposition of a few master genes. In order to understand the important factors of SINE amplification, we investigated the transcription of rat ID elements. Three different size classes of ID transcripts, BC1, BC2 and T3, have been detected in various rat tissues, including brain and testes. We have analysed the nucleotide sequences of testes- and brain-derived ID transcripts isolated by size-fractionation, C-tailing and RACE. Nucleotide sequence variation of testes ID transcripts demonstrated derivation from different loci. However, the transcripts represent a preferred set of ID elements that closely match the subfamily consensus sequences. The small ID transcripts, T3, are not comprised of primary transcripts, but are instead processed polyA-transcripts generated from many different loci. These truncated transcripts would be expected to be retroposition-incompetent forms. Therefore, the amplification of ID elements is likely to be regulated at multiple steps of retroposition, which include transcription and processing. Although brain ID transcripts showed a similar pattern, with the addition of very high levels of transcription from the BC1 locus, we also found evidence that a single locus dominated the production of brain BC2 RNA species. BC1 RNA is highly stable in both germ line and brain cells, based on the low level of detection of the processing product, T3. This stability of BC1 RNA might have been a contributing factor in its role as a master gene for ID amplification.

Rodent BC1 RNA gene as a master gene for ID element amplification.

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.

Transport of BC1 RNA in hypothalamo-neurohypophyseal axons

Ample evidence indicates that in nerve cells, several individual proteins are locally synthesized in postsynaptic domains in dendrites. By contrast, axonal terminals, at least in mammals, are generally thought to lack protein synthetic capacity. However, axonal nerve endings of the hypothalamo-neurohypophyseal tract have recently been shown to contain mRNAs encoding vasopressin, oxytocin, dynorphin, and neurofilament. In this report, we identify BC1 RNA, a small RNA polymerase III transcript that is specifically expressed in neurons, in hypothalamo-neurohypophyseal axons. BC1 RNA has previously been shown to be located in somatic and dendritic domains of various types of neurons in the rat nervous system. Here we present evidence to show that BC 1 RNA, like several neuropeptide mRNAs, is axonally transported from magnocellular hypothalamic neurons to neurosecretory nerve endings in the posterior pituitary. BC1 RNA, which has been reported to be a component of a ribonucleoprotein particle, is thus colocalized with dendritic mRNAs in dendritic domains and with axonal mRNAs in axonal domains, respectively. Such colocalization is indicative of functional interactions of BC1 RNA with those mRNAs that are targeted to extrasomatic domains of nerve cells.