Recent transcriptome analyses have shown that thousands of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. Although the number of functionally annotated ncRNAs is still limited, they are known to be frequently retained in the nucleus, where they coordinate regulatory networks of gene expression. Some subnuclear organelles or nuclear bodies include RNA species whose identity and structural roles are largely unknown. We identified 2 abundant overlapping ncRNAs, MEN and MEN␤ (MEN/␤), which are transcribed from the corresponding site in the multiple endocrine neoplasia (MEN) I locus and which localize to nuclear paraspeckles. This finding raises the intriguing possibility that MEN/␤ are involved in paraspeckle organization, because paraspeckles are, reportedly, RNase-sensitive structures. Successful removal of MEN/␤ by a refined knockdown method resulted in paraspeckle disintegration. Furthermore, the reassembly of paraspeckles disassembled by transcriptional arrest appeared to be unsuccessful in the absence of MEN/␤. RNA interference and immunoprecipitation further revealed that the paraspeckle proteins p54/nrb and PSF selectively associate with and stabilize the longer MEN␤, thereby contributing to the organization of the paraspeckle structure. The paraspeckle protein PSP1 is not directly involved in either MEN/␤ stabilization or paraspeckle organization. We postulate a model for nuclear paraspeckle body organization where specific ncRNAs and RNAbinding proteins cooperate to maintain and, presumably, establish the structure.nuclear bodies ͉ RNA-binding proteins R ecent large-scale transcriptome analyses have revealed large numbers of transcripts that do not have protein-coding potential (1, 2). Many studies have suggested that a number of long noncoding RNAs (ncRNAs) are involved in the regulation of genome organization and/or gene expression in the nucleus. Despite the identification of a handful of functional ncRNAs, including Xist, SRA, Air, and HOTAIR (3-6), the exact functions of the recently identified polyadenylated ncRNAs remain in dispute.The nucleus consists of many nuclear bodies in addition to nonrandomly arranged chromosomes (7-9). These nuclear bodies are membraneless suborganelles characterized by a distinct set of resident proteins, which provokes the question of how these compartments are assembled and maintained. There are 2 possibilities: First, an unidentified scaffold serves as an organizing center or second, the nuclear bodies are self-organized by transient interactions among their constituents. In addition to protein components, a number of RNA species reside in distinct nuclear structures, including the nucleolus (rRNA and snoRNA), the Cajal body (scaRNA and U-snRNA), and the nuclear stress bodies (satellite III RNAs) (10, 11). However, the structural role of the RNA molecule(s) in these nuclear subcompartments has not been fully investigated.We hypothesized that some of the newly discovered ncRNAs may be involved in nuclear processes in the context of nuclear bodies, and s...

Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs.

The NMDA (N-methyl-D-aspartate) subclass of glutamate receptor is essential for the synaptic plasticity thought to underlie learning and memory and for synaptic refinement during development. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or chi-1), which is expressed primarily during brain development. NR3A co-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.

Individual glomeruli in the mammalian olfactory bulb represent a single or a few type(s) of odorant receptors. Signals from different types of receptors are thus sorted out into different glomeruli. How does the neuronal circuit in the olfactory bulb contribute to the combination and integration of signals received by different glomeruli? Here we examined electrophysiologically whether there were functional interactions between mitral/tufted cells associated with different glomeruli in the rabbit olfactory bulb. First, we made simultaneous recordings of extracellular single-unit spike responses of mitral/tufted cells and oscillatory local field potentials in the dorsomedial fatty acid-responsive region of the olfactory bulb in urethan-anesthetized rabbits. Using periodic artificial inhalation, the olfactory epithelium was stimulated with a homologous series of n-fatty acids or n-aliphatic aldehydes. The odor-evoked spike discharges of mitral/tufted cells tended to phase-lock to the oscillatory local field potential, suggesting that spike discharges of many cells occur synchronously during odor stimulation. We then made simultaneous recordings of spike discharges from pairs of mitral/tufted cells located 300-500 microm apart and performed a cross-correlation analysis of their spike responses to odor stimulation. In approximately 27% of cell pairs examined, two cells with distinct molecular receptive ranges showed synchronized oscillatory discharges when olfactory epithelium was stimulated with one or a mixture of odorant(s) effective in activating both. The results suggest that the neuronal circuit in the olfactory bulb causes synchronized spike discharges of specific pairs of mitral/tufted cells associated with different glomeruli and the synchronization of odor-evoked spike discharges may contribute to the temporal binding of signals derived from different types of odorant receptor.