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...

PIWI-interacting RNAs (piRNAs) silence retrotransposons in Drosophila germ lines by associating with the PIWI proteins Argonaute 3 (AGO3), Aubergine (Aub) and Piwi. piRNAs in Drosophila are produced from intergenic repetitive genes and piRNA clusters by two systems: the primary processing pathway and the amplification loop. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, primary piRNA processing remains elusive. Here we analysed piRNA processing in a Drosophila ovarian somatic cell line where Piwi, but not Aub or AGO3, is expressed; thus, only the primary piRNAs exist. In addition to flamenco, a Piwi-specific piRNA cluster, traffic jam (tj), a large Maf gene, was determined as a new piRNA cluster. piRNAs arising from tj correspond to the untranslated regions of tj messenger RNA and are sense-oriented. piRNA loading on to Piwi may occur in the cytoplasm. zucchini, a gene encoding a putative cytoplasmic nuclease, is required for tj-derived piRNA production. In tj and piwi mutant ovaries, somatic cells fail to intermingle with germ cells and Fasciclin III is overexpressed. Loss of tj abolishes Piwi expression in gonadal somatic cells. Thus, in gonadal somatic cells, tj gives rise simultaneously to two different molecules: the TJ protein, which activates Piwi expression, and piRNAs, which define the Piwi targets for silencing.

Supporting information and the CentroidFold software are available online at: http://www.ncrna.org/software/centroidfold/.

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domaincontaining proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.

The CentroidFold web server (http://www.ncrna.org/centroidfold/) is a web application for RNA secondary structure prediction powered by one of the most accurate prediction engine. The server accepts two kinds of sequence data: a single RNA sequence and a multiple alignment of RNA sequences. It responses with a prediction result shown as a popular base-pair notation and a graph representation. PDF version of the graph representation is also available. For a multiple alignment sequence, the server predicts a common secondary structure. Usage of the server is quite simple. You can paste a single RNA sequence (FASTA or plain sequence text) or a multiple alignment (CLUSTAL-W format) into the textarea then click on the ‘execute CentroidFold’ button. The server quickly responses with a prediction result. The major advantage of this server is that it employs our original CentroidFold software as its prediction engine which scores the best accuracy in our benchmark results. Our web server is freely available with no login requirement.