Phylogenetic and Molecular Analyses Identify SNORD116 Targets Involved in the Prader–Willi Syndrome

Abstract

The eutherian-specific SNORD116 family of repeated box C/D snoRNA genes is suspected to play a major role in the Prader Willi syndrome (PWS), yet its molecular function remains poorly understood. Here, we combined phylogenetic and molecular analyses to identify candidate RNA targets. Based on the analysis of several eutherian orthologs, we found evidence of extensive birth-and-death and conversion events during SNORD116 gene history. However, the consequences for phylogenetic conservation were heterogeneous along the gene sequence. The standard snoRNA elements necessary for RNA stability and association with dedicated core proteins were the most conserved, in agreement with the hypothesis that SNORD116 generate genuine snoRNAs. Also, one of the two antisense elements (ASEs) typically involved in RNA target recognition was largely dominated by a unique sequence present in at least one subset of gene paralogs in most species, likely the result of a selective effect. In agreement with a functional role, this ASE exhibited a hybridization capacity with putative mRNA targets that was strongly conserved in Eutherians. Moreover, transient downregulation experiments in human cells showed that Snord116 controls the expression and splicing levels of these mRNAs. The functions of two of them, diacylglycerol kinase kappa (Dgkk) and Neuroligin 3 (Nlgn3), extend the description of the molecular bases of PWS and reveal unexpected molecular links with the Fragile X syndrome and autism spectrum disorders.

Dietary Conjugated Linoleic Acid Reduces Body Weight and Fat in Snord116m+/p− and Snord116m−/p− Mouse Models of Prader–Willi Syndrome

“…Interestingly, one gene was "rescued" by CLA treatment in PWS mice, Mael, in that it is expressed in WT mice, and not in PWS except when they are treated by CLA. Additionally, the Dgkk gene was found to be overexpressed in PWS, consistent with recent in vitro findings [45]. However, these findings in Mael and Dgkk have yet to be confirmed by RT-QPCR.…”

Reference Genes across Nine Brain Areas of Wild Type and Prader-Willi Syndrome Mice: Assessing Differences in Igfbp7, Pcsk1, Nhlh2 and Nlgn3 Expression

“…The second assay was specific for an isoform that includes exon 3 (Nlgn3 isoform 1, Figure 4 A,C) and the third was designed for isoforms lacking exon 3 (Nlgn3 isoform 2, Figure 4 A,D). In accordance with the previous results in cell culture [ 45 ], elimination of Snord116 cluster in PWScr m+/p− mice resulted in a slight (10–15%) increase of total Nlgn3 mRNA level in all brain regions except pons, although the difference only reached statistical significance in the hypothalamus ( Figure 4 B). Regardless of genotype, Nlgn3 was highly expressed in all brain regions, with the highest mRNA level being detected in the hippocampus, isocortex, olfactory bulb, and hypothalamus and the lowest in the cerebellum and medulla.…”

“…Subsequently, we analyzed the differential expression of Igfbp7, Nlgn3 , Pcsk1 , Pcsk2 , and Nhlh2 genes in different brain regions of WT and PWScr mice. Dysregulation of these genes in PWS model systems has been reported previously [ 13 , 45 ]. Moreover, due to the base-complementarity of SNORD116 to NHLH2 and NLGN3 mRNAs regions, the snoRNA was predicted to be involved in the posttranscriptional regulation of these genes.…”

“…The Snord116 targeting region on Nlgn3 is evolutionary conserved in mammals, including humans and mice, and located in close proximity to the intron 2–exon 3 junction, overlapping a predicted exon splice enhancer. The hybridization energy of the Snord116–Nlgn3 base–pair interaction is about −20 kcal/mol, which is similar to that of regular C/D Box snoRNA-rRNA targeting [ 45 ]. In humans, the NLGN3 gene undergoes posttranscriptional regulation during alternative splicing resulting in several different mRNA isoforms, at least two of which contain exon 3 [ 51 ].…”

“…Interestingly, the NLGN3 splice isoform-dependent regulation of synaptic transmission in the hippocampus has been shown [ 52 ]. Recently, it was reported that Snord116 could regulate alternative splicing and increase the abundance of NLGN3 isoforms that contain exon three [ 45 ]. The experiments were performed in a human HeLa S3 cell line-based Snord116 knock-down system, where the Snord116 level was reduced by about 50%, using specific antisense oligonucleotides [ 45 ].…”