Prader-Willi locus Snord116 RNA processing requires an active endogenous allele and neuron-specific splicing by Rbfox3/NeuN

Abstract

Prader-Willi syndrome (PWS), an imprinted neurodevelopmental disorder characterized by metabolic, sleep, and neuropsychiatric features, is caused by the loss of paternal SNORD116, containing only noncoding RNAs. The primary SNORD116 transcript is processed into small nucleolar RNAs (snoRNAs), which localize to nucleoli, and their spliced host gene 116HG, which is retained at its site of transcription. While functional complementation of the SNORD116 noncoding RNAs is a desirable goal for treating PWS, the mechanistic requirements of SNORD116 RNA processing are poorly understood. Here we developed and tested a novel transgenic mouse which ubiquitously expresses Snord116 on both a wild-type and Snord116 paternal deletion (Snord116 +/-) background. Interestingly, while the Snord116 transgene was ubiquitously expressed in multiple tissues, splicing of the transgene and production of snoRNAs was limited to brain tissues. Knockdown of Rbfox3, encoding neuron-specific splicing factor NeuN, in Snord116 +/--derived neurons reduced splicing of the transgene in neurons. RNA fluorescent in situ hybridization for 116HG revealed a single significantly larger signal in transgenic mice, demonstrating colocalization of transgenic and endogenous 116HG RNAs. Similarly, significantly increased snoRNA levels were detected in transgenic neuronal nucleoli, indicating that transgenic Snord116 snoRNAs were effectively processed and localized. In contrast, neither transgenic 116HG nor snoRNAs were detectable in either non-neuronal tissues or Snord116 +/neurons. Together, these results demonstrate that exogenous expression and neuron-specific splicing of the Snord116 locus are insufficient to rescue the genetic deficiency of Snord116 paternal deletion. Elucidating the mechanisms regulating Snord116 processing and localization are essential to develop effective gene replacement therapies for PWS.

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

“…Accordingly, efforts have been made to elucidate their molecular function. Transcriptome analyses of Snord116 knockout mice have revealed hundreds of differentially expressed genes (Bochukova et al 2018;Coulson et al 2018;Pace et al 2020). Also, transient overexpression of Snord115 and/or Snord116 via artificial constructs influenced the expression level of numerous genes in cell lines (Falaleeva et al 2015).…”