Deleting Snord115 genes in mice remodels monoaminergic systems activity in the brain toward cortico-subcortical imbalances.

The neuronal-specific SNORD115 has gathered interest because its deficiency may contribute to the pathophysiology of Prader-Willi syndrome (PWS), possibly by altering post-transcriptional regulation of the gene encoding the serotonin (HTR2C) receptor. Yet, Snord115-KO mice do not resume the main symptoms of PWS, and only subtle-altered A-to-I RNA editing of Htr2c mRNAs was uncovered. Because HTR2C signaling fine-tunes the activity of monoaminergic neurons, we addressed the hypothesis that lack of Snord115 alters monoaminergic systems. We first showed that Snord115 was expressed in both monoaminergic and non-monoaminergic cells of the ventral tegmental area (VTA) and the dorsal raphe nucleus (DRN) harboring cell bodies of dopaminergic and serotonergic neurons, respectively. Measuring the tissue level of monoamines and metabolites, we found very few differences except that the content of homovanillic acid-a metabolite of dopamine-was decreased in the orbitofrontal and prefrontal cortex of Snord115-KO mice. The latter effects were, however, associated with a few changes in monoamine tissue content connectivity across the 12 sampled brain regions. Using in vivo single-cell extracellular recordings, we reported that the firing rate of VTA dopaminergic neurons and DRN serotonergic neurons was significantly increased in Snord115-KO mice. These neural circuit dysfunctions were not, however, associated with apparent defects in binge eating, conditioned place preference to cocaine, cocaine-induced hyperlocomotion or compulsive behavior. Altogether, our multiscale study shows that the absence of Snord115 impacts central monoaminergic circuits to an extent that does not elicit gross behavioral abnormalities.

Antisense pairing and SNORD13 structure guide RNA cytidine acetylation.

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. The establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. After applying comparative sequence analysis and site-directed mutagenesis to provide evidence that SNORD13 folds into three main RNA helices, we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show that mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g., pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

The microRNA cluster C19MC confers differentiation potential into trophoblast lineages upon human pluripotent stem cells.

The first cell fate commitment during mammalian development is the specification of the inner cell mass and trophectoderm. This irreversible cell fate commitment should be epigenetically regulated, but the precise mechanism is largely unknown in humans. Here, we show that naïve human embryonic stem (hES) cells can transdifferentiate into trophoblast stem (hTS) cells, but primed hES cells cannot. Our transcriptome and methylome analyses reveal that a primate-specific miRNA cluster on chromosome 19 (C19MC) is active in naïve hES cells but epigenetically silenced in primed ones. Moreover, genome and epigenome editing using CRISPR/Cas systems demonstrate that C19MC is essential for hTS cell maintenance and C19MC-reactivated primed hES cells can give rise to hTS cells. Thus, we reveal that C19MC activation confers differentiation potential into trophoblast lineages on hES cells. Our findings are fundamental to understanding the epigenetic regulation of human early development and pluripotency.

Reassessment of the involvement of Snord115 in the serotonin 2c receptor pathway in a genetically relevant mouse model.

SNORD115 has been proposed to promote the activity of serotonin (HTR2C) receptor via its ability to base pair with its pre-mRNA and regulate alternative RNA splicing and/or A-to-I RNA editing. Because SNORD115 genes are deleted in most patients with the Prader-Willi syndrome (PWS), diminished HTR2C receptor activity could contribute to the impaired emotional response and/or compulsive overeating characteristic of this disease. In order to test this appealing but never demonstrated hypothesis in vivo, we created a CRISPR/Cas9-mediated Snord115 knockout mouse. Surprisingly, we uncovered only modest region-specific alterations in Htr2c RNA editing profiles, while Htr2c alternative RNA splicing was unchanged. These subtle changes, whose functional relevance remains uncertain, were not accompanied by any discernible defects in anxio-depressive-like phenotypes. Energy balance and eating behavior were also normal, even after exposure to high-fat diet. Our study raises questions concerning the physiological role of SNORD115, notably its involvement in behavioural disturbance associated with PWS.

Developmental changes of rRNA ribose methylations in the mouse.

A sequencing-based profiling method (RiboMeth-seq) for ribose methylations was used to study methylation patterns in mouse adult tissues and during development. In contrast to previous reports based on studies of human cancer cell lines, almost all methylation sites were close to fully methylated in adult tissues. A subset of sites was differentially modified in developing tissues compared to their adult counterparts and showed clear developmental dynamics. This provides the first evidence for ribosome heterogeneity at the level of rRNA modifications during mouse development. In a prominent example, the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the Gas5 host-gene and to correlate with the methylation level of its target site at LSU-G4593. The results are discussed in the context of the specialized ribosome hypothesis.

A Chemical Signature for Cytidine Acetylation in RNA.

N4-acetylcytidine (ac4C) is a highly conserved modified RNA nucleobase whose formation is catalyzed by the disease-associated N-acetyltransferase 10 (NAT10). Here we report a sensitive chemical method to localize ac4C in RNA. Specifically, we characterize the susceptibility of ac4C to borohydride-based reduction and show this reaction can cause introduction of noncognate base pairs during reverse transcription (RT). Combining borohydride-dependent misincorporation with ac4C's known base-sensitivity provides a unique chemical signature for this modified nucleobase. We show this unique reactivity can be used to quantitatively analyze cellular RNA acetylation, study adapters responsible for ac4C targeting, and probe the timing of RNA acetylation during ribosome biogenesis. Overall, our studies provide a chemical foundation for defining an expanding landscape of cytidine acetyltransferase activity and its impact on biology and disease.

Small-RNA sequencing identifies dynamic microRNA deregulation during skeletal muscle lineage progression.

Skeletal muscle satellite cells are quiescent adult resident stem cells that activate, proliferate and differentiate to generate myofibres following injury. They harbour a robust proliferation potential and self-renewing capacity enabling lifelong muscle regeneration. Although several classes of microRNAs were shown to regulate adult myogenesis, systematic examination of stage-specific microRNAs during lineage progression from the quiescent state is lacking. Here we provide a genome-wide assessment of the expression of small RNAs during the quiescence/activation transition and differentiation by RNA-sequencing. We show that the majority of small RNAs present in quiescent, activated and differentiated muscle cells belong to the microRNA class. Furthermore, by comparing expression in distinct cell states, we report a massive and dynamic regulation of microRNAs, both in numbers and amplitude, highlighting their pivotal role in regulation of quiescence, activation and differentiation. We also identify a number of microRNAs with reliable and specific expression in quiescence including several maternally-expressed miRNAs generated at the imprinted Dlk1-Dio3 locus. Unexpectedly, the majority of class-switching miRNAs are associated with the quiescence/activation transition suggesting a poised program that is actively repressed. These data constitute a key resource for functional analyses of miRNAs in skeletal myogenesis, and more broadly, in the regulation of stem cell self-renewal and tissue homeostasis.

Imprinted MicroRNA Gene Clusters in the Evolution, Development, and Functions of Mammalian Placenta.

In mammals, the expression of a subset of microRNA (miRNA) genes is governed by genomic imprinting, an epigenetic mechanism that confers monoallelic expression in a parent-of-origin manner. Three evolutionarily distinct genomic intervals contain the vast majority of imprinted miRNA genes: the rodent-specific, paternally expressed C2MC located in intron 10 of the Sfmbt2 gene, the primate-specific, paternally expressed C19MC positioned at human Chr.19q13.4 and the eutherian-specific, maternally expressed miRNAs embedded within the imprinted Dlk1-Dio3 domains at human 14q32 (also named C14MC in humans). Interestingly, these imprinted miRNA genes form large clusters composed of many related gene copies that are co-expressed with a marked, or even exclusive, localization in the placenta. Here, we summarize our knowledge on the evolutionary, molecular, and physiological relevance of these epigenetically-regulated, recently-evolved miRNAs, by focusing on their roles in placentation and possibly also in pregnancy diseases (e.g., preeclampsia, intrauterine growth restriction, preterm birth).

Box C/D small nucleolar RNA genes and the Prader-Willi syndrome: a complex interplay.

The nucleolus of mammalian cells contains hundreds of box C/D small nucleolar RNAs (SNORDs). Through their ability to base pair with ribosomal RNA precursors, most play important roles in the synthesis and/or activity of ribosomes, either by guiding sequence-specific 2'-O-methylations or by facilitating RNA folding and cleavages. A growing number of SNORD genes with elusive functions have been discovered recently. Intriguingly, the vast majority of them are located in two large, imprinted gene clusters at human chromosome region 15q11q13 (the SNURF-SNRPN domain) and at 14q32 (the DLK1-DIO3 domain) where they are expressed, respectively, only from the paternally and maternally inherited alleles. These placental mammal-specific SNORD genes have many features of the canonical SNORDs that guide 2'-O-methylations, yet they lack obvious complementarity with ribosomal RNAs and, surprisingly, they are processed from large, tandemly repeated genes expressed preferentially in the brain. This review summarizes our understanding of the biology of these peculiar SNORD genes, focusing particularly on SNORD115 and SNORD116 in the SNURF-SNRPN domain. It examines the growing evidence that altered levels of these SNORDs and/or their host-gene transcripts may be a primary cause of Prader-Willi syndrome (PWS; a rare disorder characterized by overeating and obesity) as well as abnormalities in signaling through the 5-HT2C serotonin receptor. Finally, the hypothesis that PWS may be a ribosomopathy (ribosomal disease) is also discussed. WIREs RNA 2017, 8:e1417. doi: 10.1002/wrna.1417 For further resources related to this article, please visit the WIREs website.

Deletion of the miR-379/miR-410 gene cluster at the imprinted Dlk1-Dio3 locus enhances anxiety-related behaviour.

The brain-specific miR-379/miR-410 gene cluster at the imprinted Dlk1-Dio3 domain is implicated in several aspects of brain development and function, particularly in fine-tuning the dendritic outgrowth and spine remodelling of hippocampal neurons. Whether it might influence behaviour and memory-related processes has not yet been explored at the whole organism level. We previously reported that constitutive deletion of the miR-379/miR-410 gene cluster affects metabolic adaptation in neonatal mice. Here, we examined the role of this cluster in adult brain functions by subjecting mice with the constitutive deletion to a battery of behavioural and cognitive tests. We found that the lack of miR-379/miR-410 expression is associated with abnormal emotional responses, as demonstrated by increased anxiety-related behaviour in unfamiliar environments. In contrast, spontaneous exploration, general locomotion, mood levels and sociability remained unaltered. Surprisingly, miR-379/miR-410-deficient mice also showed normal learning and spatial (or contextual) memory abilities in hippocampus-dependent tasks involving neuronal plasticity. Taken together, the imprinted miR-379/miR-410 gene cluster thus emerges as a novel regulator of the two main post-natal physiological processes previously associated with imprinted, protein-coding genes: behaviour and energy homeostasis.

Highly restricted deletion of the SNORD116 region is implicated in Prader-Willi Syndrome.

The SNORD116 locus lies in the 15q11-13 region of paternally expressed genes implicated in Prader-Willi Syndrome (PWS), a complex disease accompanied by obesity and severe neurobehavioural disturbances. Cases of PWS patients with a deletion encompassing the SNORD116 gene cluster, but preserving the expression of flanking genes, have been described. We report a 23-year-old woman who presented clinical criteria of PWS, including the behavioural and nutritional features, obesity, developmental delay and endocrine dysfunctions with hyperghrelinemia. We found a paternally transmitted highly restricted deletion of the SNORD116 gene cluster, the shortest described to date (118 kb). This deletion was also present in the father. This finding in a human case strongly supports the current hypothesis that lack of the paternal SNORD116 gene cluster has a determinant role in the pathogenesis of PWS. Moreover, targeted analysis of the SNORD116 gene cluster, complementary to SNRPN methylation analysis, should be carried out in subjects with a phenotype suggestive of PWS.

The miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain controls neonatal metabolic adaptation.

In mammals, birth entails complex metabolic adjustments essential for neonatal survival. Using a mouse knockout model, we identify crucial biological roles for the miR-379/miR-410 cluster within the imprinted Dlk1-Dio3 region during this metabolic transition. The miR-379/miR-410 locus, also named C14MC in humans, is the largest known placental mammal-specific miRNA cluster, whose 39 miRNA genes are expressed only from the maternal allele. We found that heterozygote pups with a maternal--but not paternal--deletion of the miRNA cluster display partially penetrant neonatal lethality with defects in the maintenance of energy homeostasis. This maladaptive metabolic response is caused, at least in part, by profound changes in the activation of the neonatal hepatic gene expression program, pointing to as yet unidentified regulatory pathways that govern this crucial metabolic transition in the newborn's liver. Not only does our study highlight the physiological importance of miRNA genes that recently evolved in placental mammal lineages but it also unveils additional layers of RNA-mediated gene regulation at the Dlk1-Dio3 domain that impose parent-of-origin effects on metabolic control at birth and have likely contributed to mammal evolution.

Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease.

More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease.

The SNORD115 (H/MBII-52) and SNORD116 (H/MBII-85) gene clusters at the imprinted Prader-Willi locus generate canonical box C/D snoRNAs.

The imprinted Snurf-Snrpn chromosomal domain contains two large arrays of tandemly repeated, paternally expressed box C/D small-nucleolar RNA (snoRNA) genes: the SNORD115 (H/MBII-52) and SNORD116 (H/MBII-85) gene clusters believed to play key roles in the fine-tuning of serotonin receptor (5-HT2C) pre-mRNA processing and in the etiology of the Prader-Willi Syndrome (PWS), respectively. SNORD115 and SNORD116 were recently proposed to undergo significant conversion into shorter RNA species, the so-called psnoRNAs. Here, we provide evidence that argues against the existence of abundant psnoRNAs in human or mouse brain. Instead, we characterize a previously unsuspected low-abundance, fibrillarin-associated SNORD115-derived smaller RNA species. Based on these findings, we strongly recommend that PWS-encoded SNORD115 and SNORD116 be considered as bona fide box C/D snoRNAs.

Microprocessor dynamics and interactions at endogenous imprinted C19MC microRNA genes.

Nuclear primary microRNA (pri-miRNA) processing catalyzed by the DGCR8-Drosha (Microprocessor) complex is highly regulated. Little is known, however, about how microRNA biogenesis is spatially organized within the mammalian nucleus. Here, we image for the first time, in living cells and at the level of a single microRNA cluster, the intranuclear distribution of untagged, endogenously-expressed pri-miRNAs generated at the human imprinted chromosome 19 microRNA cluster (C19MC), from the environment of transcription sites to single molecules of fully released DGCR8-bound pri-miRNAs dispersed throughout the nucleoplasm. We report that a large fraction of Microprocessor concentrates onto unspliced C19MC pri-miRNA deposited in close proximity to their genes. Our live-cell imaging studies provide direct visual evidence that DGCR8 and Drosha are targeted post-transcriptionally to C19MC pri-miRNAs as a preformed complex but dissociate separately. These dynamics support the view that, upon pri-miRNA loading and most probably concomitantly with Drosha-mediated cleavages, Microprocessor undergoes conformational changes that trigger the release of Drosha while DGCR8 remains stably bound to pri-miRNA.

Do repeated arrays of regulatory small-RNA genes elicit genomic imprinting?: Concurrent emergence of large clusters of small non-coding RNAs and genomic imprinting at four evolutionarily distinct eutherian chromosomal loci.

The basic premise of the host-defense theory is that genomic imprinting, the parent-of-origin expression of a subset of mammalian genes, derives from mechanisms originally dedicated to silencing repeated and retroviral-like sequences that deeply colonized mammalian genomes. We propose that large clusters of tandemly-repeated C/D-box small nucleolar RNAs (snoRNAs) or microRNAs represent a novel category of sequences recognized as "genomic parasites", contributing to the emergence of genomic imprinting in a subset of chromosomal regions that contain them. Such a view is supported by evidence derived from studies of the imprinted snoRNA- and/or miRNA-encoding Dlk1-Dio3, Snurf-Snrpn, Sfbmt2, and C19MC domains. While adding a new piece to the challenging puzzle of mammalian genome history, this hypothesis also reinforces the notion that dissecting the features and molecular mechanisms that discriminate between "foreign" and "endogenous" sequences is of crucial importance in the field of mammalian epigenetics.

MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression.

Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK(+)) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK(+) ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK(+) ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.

The primate-specific microRNA gene cluster (C19MC) is imprinted in the placenta.

Imprinted genes play crucial roles in mammalian development and disruption of their expression is associated with many human disorders including tumourigenesis; yet, the actual number of imprinted genes in the human genome remains a matter of debate. Here, we report on the unexpected finding that the chromosome 19 microRNA cluster (C19MC), the largest human microRNA gene cluster discovered so far, is regulated by genomic imprinting with only the paternally inherited allele being expressed in the placenta. DNA methylation profiling identified a differentially methylated region (C19MC-DMR1) that overlaps an upstream CpG-rich promoter region associated with short tandem repeats. It displays a maternal-specific methylation imprint acquired in oocytes and generates a complex population of large, compartimentalized non-coding RNA (ncRNA) species retained in close proximity to the C19MC transcription site. This occurs adjacent to, but not within, a poorly characterized nuclear Alu-rich domain. Interestingly, C19MC maps near another imprinted gene, the maternally expressed ZNF331 gene, and therefore may define a novel, previously unrecognized large imprinted primate-specific chromosomal domain. Altogether, our study adds C19MC to the growing list of imprinted repeated small RNA gene clusters and further strengthens the potential involvement of small ncRNAs in the function and/or the evolution of imprinted gene networks.