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Puralpha has been implicated in mRNA transport and translation in neurons. We previously reported that Puralpha is a component of mRNA/protein complexes (Puralpha-mRNPs) with several other proteins. Among them, we found the C9orf10 (Homo sapiens chromosome 9 open reading frame 10) protein, which was recently characterized as a component of RNA-containing structures. However, C9orf10 itself remains poorly understood. To characterize C9orf10 expression at the protein level, we raised an antibody against C9orf10 and compared the spatial and developmental expressions of this protein and Puralpha in the mouse brain. C9orf10 was expressed as early as embryo stage 12, whereas Puralpha was expressed from 5 days after birth. In adults, C9orf10 expression was most prominent in the hippocampus, caudate putamen, cerebral cortex, and cerebellum, unlike the uniform distribution of Puralpha. C9orf10-positive cells also showed immunoreactivity to Puralpha. C9orf10 expression was restricted to neurons, judging by the immunoreactivity to neuron-specific nuclear protein or CaM kinase II. These observations suggest an accessory role of C9orf10 for Puralpha in a limited brain region in addition to other possible functions that have not yet been determined.

An inverted CCAAT sequence is recognized by both transcription factors NF-Y and DNA/RNA binding protein YB-1. In the process of examining the effect of nuclear RNA on an inverted CCAAT-containing promoter of MDR1 gene, we found that U7 snRNA inhibits NF-Y and suppresses the promoter activity both in vitro and in NG108-15 tumor cells. Analysis using a designed RNA, which was structurally unrelated to U7 snRNA, revealed that RNA binding by YB-1 is not specific and that the protein is not involved in the transcription. Furthermore, we demonstrated that in the nucleus of doxorubicin-treated cells, DNA binding by NF-Y and transcriptional activity of the promoter were inhibited without either a decrease of NF-Y or an increase of the p53 tumor suppressor, which is known to inhibit DNA binding by NF-Y. In these cells, U7 snRNA was specifically increased and associated with NF-Y, and treatment with RNase A eliminated the inhibition of NF-Y activity. These results suggest that U7 snRNA has a novel function as a transcriptional regulator to control NF-Y.

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 beta-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.

Y-box proteins are DNA- and RNA-binding proteins and control specific gene expression at both transcriptional and translational levels. Particularly in germ cells, it has been reported that Y-box proteins bind to paternal or maternal mRNAs to form mRNPs, mask them from translation and control cell maturation. In this study, we cloned cDNA for a Y-box protein from rat brain. A deduced amino acid sequence of the protein was very similar to that of several other Y-box proteins, and we termed the protein rBYB1 (rat brain Y-box protein 1). rBYB1 was found to be considerably expressed in the cytoplasm of pre- and early postnatal brains, and then decreased to adult levels with brain development. Further, we found rBYB1 to be distributed in both polyribosomal and nonpolyribosomal (mRNP) fractions on a sucrose density gradient, and to be associated with polyribosomes via RNA in the higher-density fractions. Moreover, rBYB1 was localized in dendrites of the primary hippocampal neurons. We compared these sucrose gradient and intracellular rBYB1 localization results with those for fragile X mental retardation protein (FMRP), which is known to be an mRNA-binding and polyribosome-associating translational regulator distributed in neuronal dendrites. Our results suggest that in the brain of prenatal and newborn animals, rBYB1 may function in storage and/or translational regulation of mRNAs involved in the rapid progress of the postnatal brain, and in mature neurons, it may also participate in the control of protein synthesis in dendrites.

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.

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.