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...

Human centromeres consist of repetitive sequences from which satellite I noncoding RNAs are transcribed. We found that knockdown of satellite I RNA causes abnormal chromosome segregation and generation of nuclei with a grape-shape phenotype. Co-immunoprecipitation experiments showed that satellite I RNA associates with Aurora B, a component of the chromosome passenger complex (CPC) regulating proper attachment of microtubules to kinetochores, in mitotic HeLa cells. Satellite I RNA was also shown to associate with INCENP, another component of the CPC. In addition, depletion of satellite I RNA resulted in up-regulation of kinase activity of Aurora B and delocalization of the CPC from the centromere region. These results suggest that satellite I RNA is involved in chromosome segregation through controlling activity and centromeric localization of Aurora B kinase.

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint. Here, we identify the splicing factor responsible for coupling snoRNP assembly to intron excision. Intron binding protein (IBP) 160, a helicase-like protein previously detected in the spliceosomal C1 complex, binds the pre-mRNA in a sequence-independent manner, contacting nucleotides 33-40 upstream of the intron branch site, regardless of whether a snoRNA is present. Depletion of IBP160 abrogates snoRNP assembly in vitro. IBP160 binding directly to a snoRNA located too close to the intron branch site interferes with snoRNP assembly. Thus, IBP160 is the key factor linking snoRNP biogenesis and perhaps other postsplicing events to pre-mRNA splicing.

Recent large-scale transcriptome analyses have revealed that large numbers of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. They include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and longer ncRNAs, many of which are localized to the nucleus, but which have remained functionally elusive. Since ncRNAs are only known to exist in mammalian species, established experimental systems, including the Xenopus oocyte system and yeast genetics, are not available for functional analysis. RNA interference (RNAi), commonly used for analysis of protein-coding genes, is effective in eliminating cytoplasmic mRNAs, but not nuclear RNAs. To circumvent this problem, we have refined the system for knockdown of nuclear ncRNAs with chemically modified chimeric antisense oligonucleotides (ASO) that were efficiently introduced into the nucleus by nucleofection. Under optimized conditions, our system appeared to degrade at least 20 different nuclear ncRNA species in multiple mammalian cell lines with high efficiency and specificity. We also confirmed that our method had greatly improved knockdown efficiency compared with that of the previously reported method in which ASOs are introduced with transfection reagents. Furthermore, we have confirmed the expected phenotypic alterations following knockdown of HBII295 snoRNA and U7 snRNA, which resulted in a loss of site-specific methylation of the artificial RNA and the appearance of abnormal polyadenylated histone mRNA species with a concomitant delay of the cell cycle S phase, respectively. In summary, we believe that our system is a powerful tool to explore the biological functions of the large number of nuclear ncRNAs with unknown function.