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.

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.

The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with aggressive phenotype. The estrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα specific non coding transcriptome signature. Amongst putatively ERα-regulated intergenic long non coding RNAs (lncRNAs), we identified Nuclear Enriched Abundant Transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favor transcription.

Paraspeckles are subnuclear structures formed around NEAT1 lncRNA. Paraspeckles became enlarged after proteasome inhibition caused by NEAT1 transcriptional activation, leading to protein sequestration into paraspeckles. The NEAT1-dependent sequestration affects the transcription of several genes, arguing for a novel role for lncRNA in gene regulation.