While thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem cells (ESC) and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ESC regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ESCs and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ESC state.

We have developed a sensitive, accurate, and multiplexed microRNA (miRNA) profiling assay that is based on a highly efficient labeling method and novel microarray probe design. The probes provide both sequence and size discrimination, yielding in most cases highly specific detection of closely related mature miRNAs. Using a simple, single-vial experimental protocol, 120 ng of total RNA is directly labeled using Cy3 or Cy5, without fractionation or amplification, to produce precise and accurate measurements that span a linear dynamic range from 0.2 amol to 2 fmol of input miRNA. The results can provide quantitative estimates of the miRNA content for the tissues studied. The assay is also suitable for use with formalin-fixed paraffin-embedded clinical samples. Our method allows rapid design and validation of probes for simultaneous quantitative measurements of all human miRNA sequences in the public databases and to new miRNA sequences as they are reported.

Unlike mammalian and yeast cells, little is known about how plants regulate G 1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G 1 control in Saccharomyces cerevisiae does not utilize an RB-like protein.We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G 1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.Progression through the G 1 phase of the eukaryotic cell cycle is tightly regulated, allowing cells to integrate internal and external cues before initiating DNA replication and committing to a round of cell division. This process is governed by both positive-and negative-acting regulatory factors. Although substantial progress has been made in understanding the mechanisms that govern these events in yeast and mammals (reviewed in reference 61), relatively little is known about G 1 regulation in plants. The existence of cyclin-dependent kinases (Cdks) and their associated cyclin subunits in plants (reviewed in reference 14) suggests that at least some of the basic mechanisms which regulate the cell cycle have been conserved throughout eukaryotic evolution. However, identification of additional regulatory components of the plant cell cycle is clearly essential for understanding plant growth and development.In the yeast Saccharomyces cerevisiae, progression through the G 1 phase is regulated by the Cdk Cdc28 (50), which in conjunction with G 1 cyclins activates the heterodimeric Swi4/ Swi6 transcription factor (40), resulting in the transcriptional activation of genes necessary for G 1 progression and S-phase entry (12, 61). In mammalian cells, G 1 progression also depends upon a Cdk-cyclin-activated transcriptional control pathway. However, a major regulatory protein in this pathway, the retinoblastoma protein (RB), has not been found in yeast. RB is the 110-kDa product of the retinoblastoma susceptibility tumor suppressor gene and plays key roles in regulating both cell cycle progression through the G 1 ph...

Prader–Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of non-coding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple SNORD116 small nucleolar RNAs and the spliced exons of the host gene, 116HG. However, both the molecular function and the disease relevance of the spliced lncRNA 116HG are unknown. Here, we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription and increases in size in post-natal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to the dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1 and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates the diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AScausing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuronspecific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.rader-Willi syndrome (PWS) and Angelman syndrome (AS) are imprinted neurodevelopmental disorders caused by oppositely inherited deficiencies of chromosome 15q11-q13. AS and PWS are both characterized by hypotonia at birth, disordered sleep, autistic features, and intellectual disabilities, but the diseases differentiate into phenotypically distinct syndromes in early childhood (1, 2). Seizures, ataxia, and inappropriate laughter characterize AS, whereas hyperphagia leading to obesity and obsessive-compulsive behaviors characterize PWS. Maternal mutations in UBE3A/Ube3a in humans and mice have identified the loss of function of this ubiquitin E3 ligase encoding gene as the cause of AS (3, 4). For PWS, small deletions of the HBII-85/ SNORD116 locus (5-7) and two mouse models of Snord116 deletions (8, 9) have identified the minimal causative deficiency to be the paternally expressed, highly repetitive, long noncoding RNA (lncRNA) that is processed into multiple small nucleolar RNAs (snoRNAs) and spliced nuclear retained host genes (116HG and 115HG) (10,11).A recent drug screen discovered that topoisomerase inhibitors, including topotecan, reduce Ube3a-ATS by an unknown mechanism to reverse the silencing of paternal Ube3a in mouse neurons and brain (12). Topotec...