We recently showed that the mammalian genome encodes >1,000 large intergenic noncoding (linc)RNAs that are clearly conserved across mammals and, thus, functional. Gene expression patterns have implicated these lincRNAs in diverse biological processes, including cell-cycle regulation, immune surveillance, and embryonic stem cell pluripotency. However, the mechanism by which these lincRNAs function is unknown. Here, we expand the catalog of human lincRNAs to Ϸ3,300 by analyzing chromatin-state maps of various human cell types. Inspired by the observation that the well-characterized lincRNA HOTAIR binds the polycomb repressive complex (PRC)2, we tested whether many lincRNAs are physically associated with PRC2. Remarkably, we observe that Ϸ20% of lincRNAs expressed in various cell types are bound by PRC2, and that additional lincRNAs are bound by other chromatin-modifying complexes. Also, we show that siRNAmediated depletion of certain lincRNAs associated with PRC2 leads to changes in gene expression, and that the up-regulated genes are enriched for those normally silenced by PRC2. We propose a model in which some lincRNAs guide chromatin-modifying complexes to specific genomic loci to regulate gene expression.histone modifications ͉ epigenetic regulation ͉ polycomb

Summary Recently, more than a thousand large intergenic non-coding RNAs (lincRNAs) have been reported. These RNAs are evolutionarily conserved in mammalian genomes and thus presumably function in diverse biological processes. Here, we report the identification of lincRNAs that are regulated by p53. One of these lincRNAs (lincRNA-p21) serves as a repressor in p53-dependent transcriptional responses. Inhibition of lincRNA-p21 affects the expression of hundreds of gene targets enriched for genes normally repressed by p53. The observed transcriptional repression by lincRNA-p21 is mediated through the physical association with hnRNP-K. This interaction is required for proper genomic localization of hnRNP-K at repressed genes and regulation of p53 mediated apoptosis. We propose a model whereby transcription factors activate lincRNAs that serve as key repressors by physically associating with repressive complexes and modulating their localization to sets of previously active genes.

Recent transcriptomics efforts have revealed that numerous protein-coding messenger RNAs have natural antisense transcript partners, most of which seem to be noncoding RNAs. Here we identify a conserved noncoding antisense transcript for β-secretase-1 (BACE1), a crucial enzyme in Alzheimer's disease pathophysiology. The BACE1-antisense transcript (BACE1-AS) regulates BACE1 mRNA and subsequently BACE1 protein expression in vitro and in vivo. It seems that the argument for concordant regulation can only be made in the experiments with the siRNA against BACE1-AS. This convention has been followed throughout the manuscript. Please check carefully.]. Upon exposure to various cell stressors including amyloid-β 1-42 (Aβ 1-42), expression of BACE1-AS becomes elevated, increasing BACE1 mRNA stability and generating additional Aβ 1-42 through a post-transcriptional feed-forward mechanism. BACE1-AS concentrations were elevated in subjects with Alzheimer's disease as well as in amyloid precursor protein transgenic mice. These data show that BACE1 mRNA expression is under the control of a regulatory noncoding RNA that may drive Alzheimer's disease-associated pathophysiology. In summary, we report that a long noncoding RNA is directly implicated in the increased abundance of Aβ 1-42 in Alzheimer's disease.Sequential cleavage of amyloid precursor protein (APP) by BACE1, the β-site cleaving enzyme essential for Aβ 1-42 and amyloid-β 1-40 (Aβ 1-40) biosynthesis 1 , and γ-secretase NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptinitiates the 'amyloid cascade' that is central to Alzheimer's disease pathophysiology2 , 3. Oligomers of Aβ 1-42 produced by BACE1 influence key aspects of Alzheimer's disease4 -9 . Studies have revealed elevated brain BACE1 concentrations in subjects with Alzheimer's disease compared with normal controls [10][11][12][13][14][15] . However, controversy exists concerning the extent of BACE1 upregulation and whether this upregulation involves BACE1 mRNA or protein [16][17][18] .Loss of BACE1 results in numerous behavioral and physiological deficits, including memory loss 19 , emotional deficits 20 , myelination defects in peripheral nerves 21,22 and loss of synaptic plasticity 20 . Thus, the subtle but crucial boundaries between BACE1 physiology and pathology indicate that BACE1 expression must be tightly regulated, allowing the enzyme to perform its physiological functions while avoiding the serious consequences of over-or underexpression.Here we report that BACE1-AS, a natural antisense transcript, plays a part in determining BACE1 expression. BACE1-AS rapidly and reversibly upregulates BACE1 levels in response to a variety of stresses, including Aβ 1-42 exposure. Furthermore, we show elevated BACE1-AS in several brain regions of individuals with Alzheimer's disease. These data suggest that this previously unexamined noncoding RNA has a role in regulating BACE1 and in driving Alzheimer's disease pathology. RESULTS Identification of BACE1 natural antisense transcriptBACE1...

The recent discovery that the human and other mammalian genomes produce thousands of long non-coding RNAs (lncRNAs) raises many fascinating questions. These mRNA-like molecules, which lack significant protein-coding capacity, have been implicated in a wide range of biological functions through diverse and as yet poorly understood molecular mechanisms. Despite some recent insights into how lncRNAs function in such diverse cellular processes as regulation of gene expression and assembly of cellular structures, by and large, the key questions regarding lncRNA mechanisms remain to be answered. In this review, we discuss recent advances in understanding the biology of lncRNAs and propose avenues of investigation that may lead to fundamental new insights into their functions and mechanisms of action. Finally, as numerous lncRNAs are dysregulated in human diseases and disorders, we also discuss potential roles for these molecules in human health.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequence-specific posttranscriptional regulators of gene expression. Although major components of the RNA interference (RNAi) pathway have been identified, regulatory mechanisms for this pathway remain largely unknown. Here we demonstrate that the RNAi pathway can be modulated intracellularly by small molecules. We have developed a cell-based assay to monitor the activity of the RNAi pathway and find that the small-molecule enoxacin (Penetrex) enhances siRNA-mediated mRNA degradation and promotes the biogenesis of endogenous miRNAs. We show that this RNAi-enhancing activity depends on the trans-activation-responsive region RNA-binding protein. Our results provide a proofof-principle demonstration that small molecules can be used to modulate the activity of the RNAi pathway. RNAi enhancers may be useful in the development of research tools and therapeutics.

Based on the formation of the XY body at pachytene and expression studies of a few X-linked genes, the X and Y chromosomes seem to undergo transcriptional inactivation during mammalian spermatogenesis. However, the extent and the mechanism of X and Y inactivation are not known. Here, we show that both the X and Y chromosomes undergo sequential changes in their histone modifications beginning at the pachytene stage of meiosis. These changes usually are associated with transcriptional inactivation in somatic cells, and they coincide with the exclusion of the phosphorylated (active) form of RNA polymerase II from the XY body. Both sex chromosomes undergo extensive deacetylation at histones H3 and H4 and (di)methylation of lysine (K)9 on histone H3; however, there are no changes in H3-K4 methylation. These changes persist even when the XY body disappears in late pachytene, and the X and Y chromosomes segregate from one another after the first meiotic division. By the spermatid stage, histone modifications of the X and Y chromosomes revert to those of active chromatin and RNA polymerase II reengages with both chromosomes. Our observations indicate that X and Y inactivation is extensive and persists even when the X and Y chromosomes are separated in secondary spermatocytes. These findings provide insights into epigenetic programming and chromatin dynamics in the male germ line.epigenetic ͉ meiosis ͉ X inactivation ͉ chromatin ͉ methylation T he process of spermatogenesis in mammals consists of the sequential stages shown in Fig. 1, starting with diploid spermatogonia and ending in haploid sperm. At the pachytene stage ( Fig. 1), when the pairing of the homologous autosomal chromosomes occurs, the X and Y chromosomes pair only at their pseudoautosomal regions and form the sex, or XY, body (1-6). In cytological preparations, the sex body is heteropycnotic, staining more intensely than the autosomes (7). Previous studies demonstrated that several genes on the X chromosome are subject to transcriptional repression during meiosis, beginning at pachytene with reactivation by the spermatid stage (8, 9). Furthermore, RNA polymerase II has been shown to be excluded from the XY body at pachytene (10, 11). However, the mechanism and even the extent of X and Y inactivation (XYi) during spermatogenesis are not known.Unlike the inactive X in female somatic cells, the promoters of housekeeping genes on the X chromosome remain unmethylated throughout spermatogenesis in both humans and mice (8,12). Xist, the X-inactive-specific transcript gene associated with X inactivation (Xi) in female cells, is transiently transcribed in the mouse testes at pachytene and repressed again in spermatids (13,14). However, Xist transcription is not essential, given that male mice with an ablated Xist gene have normal spermatogenesis (15) and XY bodies (9, 16). Thus, the mechanisms of Xi in female somatic cells and XYi during spermatogenesis appear to have fundamental differences.Recent evidence that the XY body at pachytene is enriched for histone H3-l...

Several genome-wide transcriptomics efforts have shown that a large percentage of the mammalian genome is transcribed into RNAs, however, only a small percentage (1–2%) of these RNAs is translated into proteins. Currently there is an intense interest in characterizing the function of the different classes of noncoding RNAs and their relevance to human disease. Using genomic approaches we discovered FMR4, a primate-specific noncoding RNA transcript (2.4 kb) that resides upstream and likely shares a bidirectional promoter with FMR1. FMR4 is a product of RNA polymerase II and has a similar half-life to FMR1. The CGG expansion in the 5′ UTR of FMR1 appears to affect transcription in both directions as we found FMR4, similar to FMR1, to be silenced in fragile X patients and up-regulated in premutation carriers. Knockdown of FMR4 by several siRNAs did not affect FMR1 expression, nor vice versa, suggesting that FMR4 is not a direct regulatory transcript for FMR1. However, FMR4 markedly affected human cell proliferation in vitro; siRNAs knockdown of FMR4 resulted in alterations in the cell cycle and increased apoptosis, while the overexpression of FMR4 caused an increase in cell proliferation. Collectively, our results demonstrate an antiapoptotic function of FMR4 and provide evidence that a well-studied genomic locus can show unexpected functional complexity. It cannot be excluded that altered FMR4 expression might contribute to aspects of the clinical presentation of fragile X syndrome and/or related disorders.

Summary Decapping represents a critical control point in regulating expression of protein coding genes. Here we demonstrate that decapping also modulates expression of long non-coding RNAs (lncRNAs). Specifically, levels of >100 lncRNAs in yeast are controlled by decapping and are degraded by a pathway that occurs independent of decapping regulators. We find many lncRNAs degraded by DCP2 are expressed proximal to inducible genes. Of these, we show several genes required for galactose utilization are associated with lncRNAs that have expression patterns inversely correlated with their mRNA counterpart. Moreover, decapping of these lncRNAs is critical for rapid and robust induction of GAL gene expression. Failure to destabilize a lncRNA known to exert repressive histone modifications results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping and lncRNA degradation serve a vital role in transcriptional regulation specifically at inducible genes.