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.     

Inhibitory effect of naked neural BC1 RNA or BC200 RNA on eukaryotic in vitro translation systems is reversed by poly(A)-binding protein (PABP)

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.     

Fragile X mental retardation protein (FMRP) binds specifically to the brain cytoplasmic RNAs BC1/BC200 via a novel RNA-binding motif

Fragile X mental retardation protein (FMRP), the protein responsible for the fragile X syndrome, is an RNA-binding protein involved in localization and translation of neuronal mRNAs. One of the RNAs known to interact with FMRP is the dendritic non-translatable brain cytoplasmic RNA 1 BC1 RNA that works as an adaptor molecule linking FMRP and some of its regulated mRNAs. Here, we showed that the N terminus of FMRP binds strongly and specifically to BC1 and to its potential human analog BC200. This region does not contain a motif known to specifically recognize RNA and thus constitutes a new RNA-binding motif. We further demonstrated that FMRP recognition involves the 5' stem loop of BC1 and that this is the region that exhibits complementarity to FMRP target mRNAs, raising the possibility that FMRP plays a direct role in BC1/mRNA annealing.     

Analysis of the human alpha globin upstream regulatory element (HS-40) in transgenic mice.

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.     

The BC200 RNA Gene and Its Neural Expression Are Conserved in Anthropoidea (Primates)

The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea. The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized by a significantly higher substitution rate in the examined 5′ flanking region than in the BC200 RNA coding region itself. Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5′ flanking region (∼400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that the gene encoding BC200 RNA was created at least 35–55 million years ago and its presence, mode of expression, and association with protein(s) as an RNP are under selective pressure. Received: 1 December 1997 / Accepted: 3 June 1998     

Regulatory BC1 RNA and the fragile X mental retardation protein: convergent functionality in brain

Background

BC RNAs and the fragile X mental retardation protein (FMRP) are translational repressors that have been implicated in the control of local protein synthesis at the synapse. Work with BC1 and Fmr1 animal models has revealed that phenotypical consequences resulting from the absence of either BC1 RNA or FMRP are remarkably similar. To establish functional interactions between BC1 RNA and FMRP is important for our understanding of how local protein synthesis regulates neuronal excitability.

Methodology/Principal Findings

We generated BC1−/− Fmr1−/− double knockout (dKO) mice. We examined such animals, lacking both BC1 RNA and FMRP, in comparison with single knockout (sKO) animals lacking either one repressor. Analysis of neural phenotypical output revealed that at least three attributes of brain functionality are subject to control by both BC1 RNA and FMRP: neuronal network excitability, epileptogenesis, and place learning. The severity of CA3 pyramidal cell hyperexcitability was significantly higher in BC1−/− Fmr1−/− dKO preparations than in the respective sKO preparations, as was seizure susceptibility of BC1−/− Fmr1−/− dKO animals in response to auditory stimulation. In place learning, BC1−/− Fmr1−/− dKO animals were severely impaired, in contrast to BC1−/− or Fmr1−/− sKO animals which exhibited only mild deficits.

Conclusions/Significance

Our data indicate that BC1 RNA and FMRP operate in sequential-independent fashion. They suggest that the molecular interplay between two translational repressors directly impacts brain functionality.     

Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5′ untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.     

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice.

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.     

Cloning and characterization of the human and rabbit NUDEL-oligopeptidase promoters and their negative regulation

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5'-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.     

Expression of protein kinase C genes during ontogenic development of the central nervous system.

We have analyzed the RNA expression of three protein kinase C (PKC) genes (alpha, beta, and gamma) in human and murine central nervous systems during embryonic-fetal, perinatal, and adult life. Analysis of human brain poly(A)+ RNA indicates that expression of PKC alpha and beta genes can be detected as early as 6 weeks postconception, undergoes a gradual increase until 9 weeks postconception, and reaches its highest level in the adult stage, and that the PKC gamma gene, although not expressed during embryonic and early fetal development, is abundantly expressed in the adult period. Similar developmental patterns were observed in human spinal cord and medulla oblongata. A detailed analysis of PKC gene expression during mammalian ontogeny was performed on poly(A)+ RNA from the brain cells of murine embryos at different stages of development and the brain cells of neonatal and adult mice. The ontogenetic patterns were similar to those observed for human brain. Furthermore, we observed that the expression of PKC gamma is induced in the peri- and postnatal phases. These results suggest that expression of PKC alpha, beta, and gamma genes possibly mediates the development of central neuronal functions, and expression of PKC gamma in particular may be involved in the development of peri- and postnatal functions.