Identifying key underlying regulatory networks and predicting targets of orphan C/D box SNORD116 snoRNAs in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder characterized principally by initial symptoms of neonatal hypotonia and failure-to-thrive in infancy, followed by hyperphagia and obesity. It is well established that PWS is caused by loss of paternal expression of the imprinted region on chromosome 15q11-q13. While most PWS cases exhibit megabase-scale deletions of the paternal chromosome 15q11-q13 allele, several PWS patients have been identified harboring a much smaller deletion encompassing primarily SNORD116. This finding suggests SNORD116 is a direct driver of PWS phenotypes. The SNORD116 gene cluster is composed of 30 copies of individual SNORD116 C/D box small nucleolar RNAs (snoRNAs). Many C/D box snoRNAs have been shown to guide chemical modifications of other RNA molecules, often ribosomal RNA (rRNA). However, SNORD116 snoRNAs are termed 'orphans' because no verified targets have been identified and their sequences show no significant complementarity to rRNA. It is crucial to identify the targets and functions of SNORD116 snoRNAs because all reported PWS cases lack their expression. To address this, we engineered two different deletions modelling PWS in two distinct human embryonic stem cell (hESC) lines to control for effects of genetic background. Utilizing an inducible expression system enabled quick, reproducible differentiation of these lines into neurons. Systematic comparisons of neuronal gene expression across deletion types and genetic backgrounds revealed a novel list of 42 consistently dysregulated genes. Employing the recently described computational tool snoGloBe, we discovered these dysregulated genes are significantly enriched for predicted SNORD116 targeting versus multiple control analyses. Importantly, our results showed it is critical to use multiple isogenic cell line pairs, as this eliminated many spuriously differentially expressed genes. Our results indicate a novel gene regulatory network controlled by SNORD116 is likely perturbed in PWS patients.

TET3 epigenetically controls feeding and stress response behaviors via AGRP neurons.

The TET family of dioxygenases promote DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Hypothalamic agouti-related peptide-expressing (AGRP-expressing) neurons play an essential role in driving feeding, while also modulating nonfeeding behaviors. Besides AGRP, these neurons produce neuropeptide Y (NPY) and the neurotransmitter GABA, which act in concert to stimulate food intake and decrease energy expenditure. Notably, AGRP, NPY, and GABA can also elicit anxiolytic effects. Here, we report that in adult mouse AGRP neurons, CRISPR-mediated genetic ablation of Tet3, not previously known to be involved in central control of appetite and metabolism, induced hyperphagia, obesity, and diabetes, in addition to a reduction of stress-like behaviors. TET3 deficiency activated AGRP neurons, simultaneously upregulated the expression of Agrp, Npy, and the vesicular GABA transporter Slc32a1, and impeded leptin signaling. In particular, we uncovered a dynamic association of TET3 with the Agrp promoter in response to leptin signaling, which induced 5hmC modification that was associated with a chromatin-modifying complex leading to transcription inhibition, and this regulation occurred in both the mouse models and human cells. Our results unmasked TET3 as a critical central regulator of appetite and energy metabolism and revealed its unexpected dual role in the control of feeding and other complex behaviors through AGRP neurons.

Transcriptome-Wide Identification of 2'-O-Methylation Sites with RibOxi-Seq.

The ability to detect 2'-O-methylation sites (Nm) in high-throughput fashion is important, as increasing evidence points to a more diverse landscape for this RNA modification as well as the possibility of yet unidentified functions. Here we describe an optimized version of RibOxi-seq, which is built upon the original published method, that not only accurately profiles ribosomal RNA (rRNA) Nm sites with minimal RNA input but is also robust enough to identify mRNA intronic and exonic sites.

Prader-Willi syndrome: reflections on seminal studies and future therapies.

Prader-Willi syndrome (PWS) is caused by the loss of function of the paternally inherited 15q11-q13 locus. This region is governed by genomic imprinting, a phenomenon in which genes are expressed exclusively from one parental allele. The genomic imprinting of the 15q11-q13 locus is established in the germline and is largely controlled by a bipartite imprinting centre. One part, termed the Prader-Willi syndrome imprinting center (PWS-IC), comprises a CpG island that is unmethylated on the paternal allele and methylated on the maternal allele. The second part, termed the Angelman syndrome imprinting centre, is required to silence the PWS_IC in the maternal germline. The loss of the paternal contribution of the imprinted 15q11-q13 locus most frequently occurs owing to a large deletion of the entire imprinted region but can also occur through maternal uniparental disomy or an imprinting defect. While PWS is considered a contiguous gene syndrome based on large-deletion and uniparental disomy patients, the lack of expression of only non-coding RNA transcripts from the SNURF-SNRPN/SNHG14 may be the primary cause of PWS. Patients with small atypical deletions of the paternal SNORD116 cluster alone appear to have most of the PWS related clinical phenotypes. The loss of the maternal contribution of the 15q11-q13 locus causes a separate and distinct condition called Angelman syndrome. Importantly, while much has been learned about the regulation and expression of genes and transcripts deriving from the 15q11-q13 locus, there remains much to be learned about how these genes and transcripts contribute at the molecular level to the clinical traits and developmental aspects of PWS that have been observed.

The large repertoire of 2'-O-methylation guided by C/D snoRNAs on Trypanosoma brucei rRNA.

The parasite Trypanosoma brucei cycles between insect and mammalian hosts, and is the causative agent of sleeping sickness. Here, we performed genome-wide mapping of 2'-O-methylations (Nms) on trypanosome rRNA using three high-throughput sequencing methods; RibOxi-seq, RiboMeth-seq and 2'-OMe-seq. This is the first study using three genome-wide mapping approaches on rRNA from the same species showing the discrepancy among the methods. RibOxi-seq detects all the sites, but RiboMeth-seq is the only method to evaluate the level of a single Nm site. The sequencing revealed at least ninety-nine Nms guided by eighty-five snoRNAs among these thirty-eight Nms are trypanosome specific sites. We present the sequence and target of the C/D snoRNAs guiding on rRNA. This is the highest number of Nms detected to date on rRNA of a single cell parasite. Based on RiboMeth-seq, several Nm sites were found to be differentially regulated at the two stages of the parasite life cycle, the insect procyclic form (PCF) versus the bloodstream form (BSF) in the mammalian host.

Distinct Processing of lncRNAs Contributes to Non-conserved Functions in Stem Cells.

Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.

Hepatic TET3 contributes to type-2 diabetes by inducing the HNF4α fetal isoform.

Precise control of hepatic glucose production (HGP) is pivotal to maintain systemic glucose homeostasis. HNF4α functions to stimulate transcription of key gluconeogenic genes. HNF4α harbors two promoters (P2 and P1) thought to be primarily active in fetal and adult livers, respectively. Here we report that the fetal version of HNF4α is required for HGP in the adult liver. This isoform is acutely induced upon fasting and chronically increased in type-2 diabetes (T2D). P2 isoform induction occurs in response to glucagon-stimulated upregulation of TET3, not previously shown to be involved in HGP. TET3 is recruited to the P2 promoter by FOXA2, leading to promoter demethylation and increased transcription. While TET3 overexpression augments HGP, knockdown of either TET3 or the P2 isoform alone in the liver improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies unmask an unanticipated, conserved regulatory mechanism in HGP and offer potential therapeutic targets for T2D.

Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.

Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.

CIRCexplorer3: A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression.

Sequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Therefore, examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies. Thus, direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. Here, we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CIRCexplorer3-CLEAR). A new quantitation parameter, fragments per billion mapped bases (FPB), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression level using linear RNA expression level as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation. CIRCexplorer3-CLEAR is publically available at https://github.com/YangLab/CLEAR.

H19 lncRNA Promotes Skeletal Muscle Insulin Sensitivity in Part by Targeting AMPK.

Skeletal muscle plays a pivotal role in regulating systemic glucose homeostasis in part through the conserved cellular energy sensor AMPK. AMPK activation increases glucose uptake, lipid oxidation, and mitochondrial biogenesis, leading to enhanced muscle insulin sensitivity and whole-body energy metabolism. Here we show that the muscle-enriched H19 long noncoding RNA (lncRNA) acts to enhance muscle insulin sensitivity, at least in part, by activating AMPK. We identify the atypical dual-specificity phosphatase DUSP27/DUPD1 as a potentially important downstream effector of H19. We show that DUSP27, which is highly expressed in muscle with previously unknown physiological function, interacts with and activates AMPK in muscle cells. Consistent with decreased H19 expression in the muscle of insulin-resistant human subjects and rodents, mice with genetic H19 ablation exhibit muscle insulin resistance. Furthermore, a high-fat diet downregulates muscle H19 via both posttranscriptional and epigenetic mechanisms. Our results uncover an evolutionarily conserved, highly expressed lncRNA as an important regulator of muscle insulin sensitivity.

Elevated hepatic expression of H19 long noncoding RNA contributes to diabetic hyperglycemia.

Excessive hepatic glucose production (HGP) contributes significantly to the hyperglycemia of type 2 diabetes; however, the molecular mechanism underlying this dysregulation remains poorly understood. Here, we show that fasting temporally increases the expression of H19 long noncoding RNA (lncRNA) in nondiabetic mouse liver, whereas its level is chronically elevated in diet-induced diabetic mice, consistent with the previously reported chronic hepatic H19 increase in diabetic patients. Importantly, liver-specific H19 overexpression promotes HGP, hyperglycemia, and insulin resistance, while H19 depletion enhances insulin-dependent suppression of HGP. Using genome-wide methylation and transcriptome analyses, we demonstrate that H19 knockdown in hepatic cells alters promoter methylation and expression of Hnf4a, a master gluconeogenic transcription factor, and that this regulation is recapitulated in vivo. Our findings offer a mechanistic explanation of lncRNA H19's role in the pathogenesis of diabetic hyperglycemia and suggest that targeting hepatic H19 may hold the potential of new treatment for this disease.

High-throughput and site-specific identification of 2'-O-methylation sites using ribose oxidation sequencing (RibOxi-seq).

Ribose methylation (2'-O-methylation, 2'-OMe) occurs at high frequencies in rRNAs and other small RNAs and is carried out using a shared mechanism across eukaryotes and archaea. As RNA modifications are important for ribosome maturation, and alterations in these modifications are associated with cellular defects and diseases, it is important to characterize the landscape of 2'-O-methylation. Here we report the development of a highly sensitive and accurate method for ribose methylation detection using next-generation sequencing. A key feature of this method is the generation of RNA fragments with random 3'-ends, followed by periodate oxidation of all molecules terminating in 2',3'-OH groups. This allows only RNAs harboring 2'-OMe groups at their 3'-ends to be sequenced. Although currently requiring microgram amounts of starting material, this method is robust for the analysis of rRNAs even at low sequencing depth.

An Ultraconserved Element (UCE) controls homeostatic splicing of ARGLU1 mRNA.

Arginine and Glutamate-Rich protein 1 (ARGLU1) is a protein whose function is poorly understood, but may act in both transcription and pre-mRNA splicing. We demonstrate that the ARGLU1 gene expresses at least three distinct RNA splice isoforms - a fully spliced isoform coding for the protein, an isoform containing a retained intron that is detained in the nucleus, and an isoform containing an alternative exon that targets the transcript for nonsense mediated decay. Furthermore, ARGLU1 contains a long, highly evolutionarily conserved sequence known as an Ultraconserved Element (UCE) that is within the retained intron and overlaps the alternative exon. Manipulation of the UCE, in a reporter minigene or via random mutations in the genomic context using CRISPR/Cas9, changed the splicing pattern. Further, overexpression of the ARGLU1 protein shifted the splicing of endogenous ARGLU1 mRNA, resulting in an increase in the retained intron isoform and nonsense mediated decay susceptible isoform and a decrease in the fully spliced isoform. Taken together with data showing that functional protein knockout shifts splicing toward the fully spliced isoform, our data are consistent with a model in which unproductive splicing complexes assembled at the alternative exon lead to inefficient splicing and intron retention.

Gene Regulation and Quality Control in Murine Polyomavirus Infection.

Murine polyomavirus (MPyV) infects mouse cells and is highly oncogenic in immunocompromised hosts and in other rodents. Its genome is a small, circular DNA molecule of just over 5000 base pairs and it encodes only seven polypeptides. While seemingly simply organized, this virus has adopted an unusual genome structure and some unusual uses of cellular quality control pathways that, together, allow an amazingly complex and varied pattern of gene regulation. In this review we discuss how MPyV leverages these various pathways to control its life cycle.

H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase.

DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components.

Global Analysis of Mouse Polyomavirus Infection Reveals Dynamic Regulation of Viral and Host Gene Expression and Promiscuous Viral RNA Editing.

Mouse polyomavirus (MPyV) lytically infects mouse cells, transforms rat cells in culture, and is highly oncogenic in rodents. We have used deep sequencing to follow MPyV infection of mouse NIH3T6 cells at various times after infection and analyzed both the viral and cellular transcriptomes. Alignment of sequencing reads to the viral genome illustrated the transcriptional profile of the early-to-late switch with both early-strand and late-strand RNAs being transcribed at all time points. A number of novel insights into viral gene expression emerged from these studies, including the demonstration of widespread RNA editing of viral transcripts at late times in infection. By late times in infection, 359 host genes were seen to be significantly upregulated and 857 were downregulated. Gene ontology analysis indicated transcripts involved in translation, metabolism, RNA processing, DNA methylation, and protein turnover were upregulated while transcripts involved in extracellular adhesion, cytoskeleton, zinc finger binding, SH3 domain, and GTPase activation were downregulated. The levels of a number of long noncoding RNAs were also altered. The long noncoding RNA MALAT1, which is involved in splicing speckles and used as a marker in many late-stage cancers, was noticeably downregulated, while several other abundant noncoding RNAs were strongly upregulated. We discuss these results in light of what is currently known about the MPyV life cycle and its effects on host cell growth and metabolism.

Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine.

Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine.

SnoVectors for nuclear expression of RNA.

Many long noncoding RNAs (lncRNAs) are constrained to the nucleus to exert their functions. However, commonly used vectors that were designed to express mRNAs have not been optimized for the study of nuclear RNAs. We reported recently that sno-lncRNAs are not capped or polyadenylated but rather are terminated on each end by snoRNAs and their associated proteins. These RNAs are processed from introns and are strictly confined to the nucleus. Here we have used these features to design expression vectors that can stably express virtually any sequence of interest and constrain its accumulation to the nucleus. Further, these RNAs appear to retain normal nuclear associations and function. SnoVectors should be useful in conditions where nuclear RNA function is studied or where export to the cytoplasm needs to be avoided.

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells.

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.

Long noncoding RNAs in imprinting and X chromosome inactivation.

The field of long noncoding RNA (lncRNA) research has been rapidly advancing in recent years. Technological advancements and deep-sequencing of the transcriptome have facilitated the identification of numerous new lncRNAs, many with unusual properties, however, the function of most of these molecules is still largely unknown. Some evidence suggests that several of these lncRNAs may regulate their own transcription in cis, and that of nearby genes, by recruiting remodeling factors to local chromatin. Notably, lncRNAs are known to exist at many imprinted gene clusters. Genomic imprinting is a complex and highly regulated process resulting in the monoallelic silencing of certain genes, based on the parent-of-origin of the allele. It is thought that lncRNAs may regulate many imprinted loci, however, the mechanism by which they exert such influence is poorly understood. This review will discuss what is known about the lncRNAs of major imprinted loci, and the roles they play in the regulation of imprinting.