Studies of the transcriptional output of the human and mouse genomes have revealed that there are many more transcripts produced than can be accounted for by predicted protein-coding genes. Using a custom microarray, we have identified 184 non-coding RNAs that exhibit more than twofold up-or down-regulation upon differentiation of C2C12 myoblasts into myotubes. Here, we focus on the Men e/b locus, which is up-regulated 3.3-fold during differentiation. Two non-coding RNA isoforms are produced from a single RNA polymerase II promoter, differing in the location of their 39 ends. Men e is a 3.2-kb polyadenylated RNA, whereas Men b is an ;20-kb transcript containing a genomically encoded poly(A)-rich tract at its 39-end. The 39-end of Men b is generated by RNase P cleavage. The Men e/b transcripts are localized to nuclear paraspeckles and directly interact with NONO. Knockdown of MEN e/b expression results in the disruption of nuclear paraspeckles. Furthermore, the formation of paraspeckles, after release from transcriptional inhibition by DRB treatment, was suppressed in MEN e/b-depleted cells. Our findings indicate that the MEN e/b non-coding RNAs are essential structural/organizational components of paraspeckles.

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

The past few years have revealed that the genomes of all studied eukaryotes are almost entirely transcribed, generating an enormous number of non-protein-coding RNAs (ncRNAs). In parallel, it is increasingly evident that many of these RNAs have regulatory functions. Here, we highlight recent advances that illustrate the diversity of ncRNA control of genome dynamics, cell biology, and developmental programming.

Large numbers of long RNAs with little or no protein-coding potential [long noncoding RNAs (lncRNAs)] are being identified in eukaryotes. In parallel, increasing data describing the expression profiles, molecular features and functions of individual lncRNAs in a variety of systems are accumulating. To enable the systematic compilation and updating of this information, we have developed a database (lncRNAdb) containing a comprehensive list of lncRNAs that have been shown to have, or to be associated with, biological functions in eukaryotes, as well as messenger RNAs that have regulatory roles. Each entry contains referenced information about the RNA, including sequences, structural information, genomic context, expression, subcellular localization, conservation, functional evidence and other relevant information. lncRNAdb can be searched by querying published RNA names and aliases, sequences, species and associated protein-coding genes, as well as terms contained in the annotations, such as the tissues in which the transcripts are expressed and associated diseases. In addition, lncRNAdb is linked to the UCSC Genome Browser for visualization and Noncoding RNA Expression Database (NRED) for expression information from a variety of sources. lncRNAdb provides a platform for the ongoing collation of the literature pertaining to lncRNAs and their association with other genomic elements. lncRNAdb can be accessed at: http://www.lncrnadb.org/.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease. Sections Consensus statement Purpose of this Consensus StatementIn this Consensus Statement we present a current and coherent picture of the roles of lncRNAs in cell and developmental biology, identify the key issues in understanding their functions and chart the path forward. We address lncRNA definition, nomenclature, conservation, expression, phenotypic visibility, functional assays and molecular mechanisms encompassing lncRNA connections to chromatin architecture, epigenetic processes, enhancer function and biomolecular condensates, as well as the roles of lncRNAs outside the nucleus. We argue that loci expressing lncRNAs should be recognized as bona fide genes and discuss lncRNA structure-function relationships as the means to parse mechanisms and pathways. Finally, we identify the current challenges and offer recommendations for understanding the relationship of lncRNAs to genome architecture, gene regulation and cellular organization.The authors of this Consensus Statement were suggested by recommendations of colleagues. Consensus was reached by group e-mail and discussion. Definition and nomenclature of lncRNAslncRNAs have been arbitrarily defined as non-coding transcripts of more than 200 nucleotides (200 nt), which is a convenient size cutoff in biochemical and biophysical RNA purification protocols that deplete most infrastructural RNAs, such as 5S rRNAs, tRNAs, snRNAs and snoRNAs, as well as miRNAs, siRNAs and piRNAs 23 . This definition also excludes some other well-known short RNAs such as the primatespecific snaRs (~80-120 nt), which associate with nuclear factor 90 (ref. 24); Y ...