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.

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.

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.

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.

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.

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.

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.

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.

Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene.

MicroRNAs (miRNAs) are an abundant class of RNAs that are approximately 21-25 nucleotides (nt) long, interact with mRNAs and trigger either translation repression or RNA cleavage (RNA interference, RNAi) depending on the degree of complementarity with their targets. Here we show that the imprinted mouse distal chromosome 12 locus encodes two miRNA genes expressed from the maternally inherited chromosome and antisense to a retrotransposon-like gene (Rtl1) expressed only from the paternal allele.

Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12.

Genomic imprinting causes parental origin-specific gene expression. Cis-acting regulatory elements that control imprinting are not fully understood but involve regions that become differentially methylated on the two parental chromosomes during male and female gametogenesis. Understanding properties of maternally and paternally inherited imprints provides insight into the mechanisms and evolution of genomic imprinting. Previously we identified an intergenic germline-derived differentially methylated region (IG-DMR) that is a candidate control element for an imprinted domain on distal mouse chromosome 12 (ref. 5). The 1-Mb cluster contains the paternally expressed protein-coding genes Dlk1 (refs. 6,7) and Dio3 (ref. 8,9) and several maternally expressed non-coding RNAs, including Gtl2 (refs. 6,7,10) and C/D snoRNAs. A retrotransposon-like gene (Rtl1) is expressed from the paternal chromosome and has an antisense transcript expressed from the maternal chromosome containing two microRNAs with full complementarity to Rtl1 (ref. 12). Here we show that deletion of the IG-DMR from the maternally inherited chromosome causes bidirectional loss of imprinting of all genes in the cluster. When the deletion is transmitted from the father, imprinting is unaltered. These results prove that the IG-DMR is a control element for all imprinted genes on the maternal chromosome only and indicate that the two parental chromosomes control allele-specific gene expression differently.

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.

Long nuclear-retained non-coding RNAs and allele-specific higher-order chromatin organization at imprinted snoRNA gene arrays.

The imprinted Snurf-Snrpn domain, also referred to as the Prader-Willi syndrome region, contains two approximately 100-200 kb arrays of repeated small nucleolar (sno)RNAs processed from introns of long, paternally expressed non-protein-coding RNAs whose biogenesis and functions are poorly understood. We provide evidence that C/D snoRNAs do not derive from a single transcript as previously envisaged, but rather from (at least) two independent transcription units. We show that spliced snoRNA host-gene transcripts accumulate near their transcription sites as structurally constrained RNA species that are prevented from diffusing, as well as multiple stable nucleoplasmic RNA foci dispersed in the entire nucleus but not in the nucleolus. Chromatin structure at these repeated arrays displays an outstanding parent-of-origin-specific higher-order organization: the transcriptionally active allele is revealed as extended DNA FISH signals whereas the genetically identical, silent allele is visualized as singlet DNA FISH signals. A similar allele-specific chromatin organization is documented for snoRNA gene arrays at the imprinted Dlk1-Dio3 domain. Our findings have repercussions for understanding the spatial organization of gene expression and the intra-nuclear fate of non-coding RNAs in the context of nuclear architecture.

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.

C19MC microRNAs are processed from introns of large Pol-II, non-protein-coding transcripts.

MicroRNAs are tiny RNA molecules that play important regulatory roles in a broad range of developmental, physiological or pathological processes. Despite recent progress in our understanding of miRNA processing and biological functions, little is known about the regulatory mechanisms that control their expression at the transcriptional level. C19MC is the largest human microRNA gene cluster discovered to date. This 100-kb long cluster consists of 46 tandemly repeated, primate-specific pre-miRNA genes that are flanked by Alu elements (Alus) and embedded within a approximately 400- to 700-nt long repeated unit. It has been proposed that C19MC miRNA genes are transcribed by RNA polymerase III (Pol-III) initiating from A and B boxes embedded in upstream Alu repeats. Here, we show that C19MC miRNAs are intron-encoded and processed by the DGCR8-Drosha (Microprocessor) complex from a previously unidentified, non-protein-coding Pol-II (and not Pol-III) transcript which is mainly, if not exclusively, expressed in the placenta.

ADAR2-mediated editing of RNA substrates in the nucleolus is inhibited by C/D small nucleolar RNAs.

Posttranscriptional, site-specific adenosine to inosine (A-to-I) base conversions, designated as RNA editing, play significant roles in generating diversity of gene expression. However, little is known about how and in which cellular compartments RNA editing is controlled. Interestingly, the two enzymes that catalyze RNA editing, adenosine deaminases that act on RNA (ADAR) 1 and 2, have recently been demonstrated to dynamically associate with the nucleolus. Moreover, we have identified a brain-specific small RNA, termed MBII-52, which was predicted to function as a nucleolar C/D RNA, thereby targeting an A-to-I editing site (C-site) within the 5-HT2C serotonin receptor pre-mRNA for 2'-O-methylation. Through the subcellular targeting of minigenes that contain natural editing sites, we show that ADAR2- but not ADAR1-mediated RNA editing occurs in the nucleolus. We also demonstrate that MBII-52 forms a bona fide small nucleolar ribonucleoprotein particle that specifically decreases the efficiency of RNA editing by ADAR2 at the targeted C-site. Our data are consistent with a model in which C/D small nucleolar RNA might play a role in the regulation of RNA editing.

Bsr, a nuclear-retained RNA with monoallelic expression.

The imprinted Dlk1-Gtl2 and Prader-Willi syndrome (PWS) regions are characterized by a complex noncoding transcription unit spanning arrays of tandemly repeated C/D RNA genes. These noncoding RNAs (ncRNAs) are thought to play an essential but still poorly understood role. To better understand the intracellular fate of these large ncRNAs, fluorescence in situ hybridization was carried out at the rat Dlk1-Gtl2 domain. This locus contains a approximately 100-kb-long gene cluster comprising 86 homologous RBII-36 C/D RNA gene copies, all of them intron-encoded within the ncRNA gene Bsr. Here, we demonstrate that the Bsr gene is monoallelically expressed in primary rat embryonic fibroblasts as well as in hypothalamic neurons and yields a large amount of unspliced and spliced RNAs at the transcription site, mostly as elongated RNA signals. Surprisingly, spliced Bsr RNAs released from the transcription site mainly concentrate as numerous, stable nuclear foci that do not colocalize with any known subnuclear structures. On drug treatments, a fraction of Bsr RNA relocalizes to the cytoplasm and associates with stress granules (SGs), but not with P-bodies, pointing to a potential link between SGs and the metabolism of ncRNA. Thus, Bsr might represent a novel type of nuclear-retained transcript.