SnoRNA Snord116 (Pwcr1/MBII-85) deletion causes growth deficiency and hyperphagia in mice

Prader-Willi syndrome (PWS) is the leading genetic cause of obesity. After initial severe hypotonia, PWS children become hyperphagic and morbidly obese, if intake is not restricted. Short stature with abnormal growth hormone secretion, hypogonadism, cognitive impairment, anxiety and behavior problems are other features. PWS is caused by lack of expression of imprinted genes in a approximately 4 mb region of chromosome band 15q11.2. Our previous translocation studies predicted a major role for the C/D box small nucleolar RNA cluster SNORD116 (PWCR1/HBII-85) in PWS. To test this hypothesis, we created a approximately 150 kb deletion of the > 40 copies of Snord116 (Pwcr1/MBII-85) in C57BL/6 mice. Snord116del mice with paternally derived deletion lack expression of this snoRNA. They have early-onset postnatal growth deficiency, but normal fertility and lifespan. While pituitary structure and somatotrophs are normal, liver Igf1 mRNA is decreased. In cognitive and behavior tests, Snord116del mice are deficient in motor learning and have increased anxiety. Around three months of age, they develop hyperphagia, but stay lean on regular and high-fat diet. On reduced caloric intake, Snord116del mice maintain their weight better than wild-type littermates, excluding increased energy requirement as a cause of hyperphagia. Normal compensatory feeding after fasting, and ability to maintain body temperature in the cold indicate normal energy homeostasis regulation. Metabolic chamber studies reveal that Snord116del mice maintain energy homeostasis by altered fuel usage. Prolonged mealtime and increased circulating ghrelin indicate a defect in meal termination mechanism. Snord116del mice, the first snoRNA deletion animal model, reveal a novel role for a non-coding RNA in growth and feeding regulation.     

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.     

RNA-Seq of the Nucleolus Reveals Abundant SNORD44-Derived Small RNAs

Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19–20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5′ and 3′ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha–mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.     

Identification of novel ribonucleo-protein complexes from the brain-specific snoRNA MBII-52

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.     

Deletion of the MBII-85 snoRNA gene cluster in mice results in postnatal growth retardation

Prader-Willi syndrome (PWS [MIM 176270]) is a neurogenetic disorder characterized by decreased fetal activity, muscular hypotonia, failure to thrive, short stature, obesity, mental retardation, and hypogonadotropic hypogonadism. It is caused by the loss of function of one or more imprinted, paternally expressed genes on the proximal long arm of chromosome 15. Several potential PWS mouse models involving the orthologous region on chromosome 7C exist. Based on the analysis of deletions in the mouse and gene expression in PWS patients with chromosomal translocations, a critical region (PWScr) for neonatal lethality, failure to thrive, and growth retardation was narrowed to the locus containing a cluster of neuronally expressed MBII-85 small nucleolar RNA (snoRNA) genes. Here, we report the deletion of PWScr. Mice carrying the maternally inherited allele (PWScr^m−/p+) are indistinguishable from wild-type littermates. All those with the paternally inherited allele (PWScr^m+/p−) consistently display postnatal growth retardation, with about 15% postnatal lethality in C57BL/6, but not FVB/N crosses. This is the first example in a multicellular organism of genetic deletion of a C/D box snoRNA gene resulting in a pronounced phenotype.     

The noncoding RNAs SNORD50A and SNORD50B-mediated TRIM21-GMPS interaction promotes the growth of p53 wild-type breast cancers by degrading p53

Small nucleolar RNA SNORD50A and SNORD50B (SNORD50A/B) has been reported to be recurrently deleted and function as a putative tumor suppressor in different types of cancer by binding to and suppressing the activity of the KRAS oncoproteins. Its deletion correlates with poorer patient survival. However, in this study, we surprisingly found that SNORD50A/B loss predicted a better survival in breast cancer patients carrying wild-type p53. Functional studies showed that SNORD50A/B deletion strongly inhibited the proliferation, migration, invasion and tumorigenic potential, and induced cell cycle arrest and apoptosis in p53 wild-type breast cancer cells, while exerted the opposite effects in p53 mutated breast cancer cells. This was also supported by ectopically expressing SNORD50A/B in both p53 wild-type and mutated breast cancer cells. Mechanistically, SNORD50A/B clearly enhances the interaction between E3 ubiquitin ligase TRIM21 and its substrate GMPS by forming a complex among them, thereby promoting GMPS ubiquitination and its subsequent cytoplasmic sequestration. SNORD50A/B deletion in p53 wild-type breast cancer cells will release GMPS and induce the translocation of GMPS into the nucleus, where GMPS can recruit USP7 and form a complex with p53, thereby decreasing p53 ubiquitination, stabilizing p53 proteins, and inhibiting malignant phenotypes of cancer cells. Altogether, the present study first reports that SNORD50A/B plays an oncogenic role in p53 wild-type breast cancers by mediating TRIM21-GMPS interaction.Subject terms: Cancer genetics, Tumour biomarkers     

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.     

Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea

Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent K(d) ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.     

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:     

RNA structure and function in C/D and H/ACA s(no)RNPs

From archaea to humans, C/D- and H/ACA-type small ribonucleoprotein particles play key roles in crucial RNA processing events. Various such particles are required for pre-rRNA cleavage steps and/or for chemical modification of rRNAs, spliceosomal small nuclear RNAs, tRNAs and perhaps even mRNAs. Each C/D-type particle contains a small RNA possessing conserved C and D, as well as related C' and D', sequence motifs, whereas each H/ACA-type particle contains a small RNA featuring conserved H and ACA sequence elements. Recently published studies highlight the importance of sequence and structural elements of these RNAs in the localization, activity and assembly of the ribonucleoprotein particles. A novel sequence element, the Cajal body box, found at the apex of stem structures within a subset of H/ACA small RNAs, mediates the specific retention of particles containing these elements inside nucleoplasmic Cajal bodies. Two highly conserved elements, the m1 and m2 boxes, have been identified in the 3' stem of the atypical H/ACA snR30/U17 RNAs. These conserved sequence elements are necessary for early pre-rRNA cleavage events and consequently for mature 18S rRNA production. Finally, convincing evidence has been provided that the conserved C and D sequence motifs of C/D-type small RNAs fold into a helix-bulge-helix structure, called a kink-turn, that provides a platform for assembly of C/D-type ribonucleoprotein particles.     

Identification of human miRNA precursors that resemble box C/D snoRNAs

There are two main classes of small nucleolar RNAs (snoRNAs): the box C/D snoRNAs and the box H/ACA snoRNAs that function as guide RNAs to direct sequence-specific modification of rRNA precursors and other nucleolar RNA targets. A previous computational and biochemical analysis revealed a possible evolutionary relationship between miRNA precursors and some box H/ACA snoRNAs. Here, we investigate a similar evolutionary relationship between a subset of miRNA precursors and box C/D snoRNAs. Computational analyses identified 84 intronic miRNAs that are encoded within either box C/D snoRNAs, or in precursors showing similarity to box C/D snoRNAs. Predictions of the folded structures of these box C/D snoRNA-like miRNA precursors resemble the structures of known box C/D snoRNAs, with the boxes C and D often in close proximity in the folded molecule. All five box C/D snoRNA-like miRNA precursors tested (miR-27b, miR-16-1, mir-28, miR-31 and let-7g) bind to fibrillarin, a specific protein component of functional box C/D snoRNP complexes. The data suggest that a subset of small regulatory RNAs may have evolved from box C/D snoRNAs.     

Sequential 2'-O-methylation of archaeal pre-tRNATrp nucleotides is guided by the intron-encoded but trans-acting box C/D ribonucleoprotein of pre-tRNA

Haloferax volcanii pre-tRNA(Trp) processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNA(Trp) in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNA(Trp) or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson-Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNA(Trp) added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intron-encoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pre-tRNA(Trp) intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing.     

Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs

Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited. In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p. We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other. We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.