PWS-locus encoded non-coding RNAs in Knock-In mouse models

• Applicants: Timofey Rozhdestvensky, Ph.D.; Boris Skryabin, Ph.D.
• Subject Area: Human Genetics ; General Genetics and Functional Genome Biology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2017 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 391469518

Prader-Willi syndrome (PWS) is a neurogenetic disorder caused by deletion or inactivation of imprinted genes within the PWS-locus on paternally inherited human chromosome 15. PWS was the first human disease found to be directly associated with the genomic deletion of non-protein coding RNAs (ncRNAs) or the PWS-critical region (PWScr). The PWScr contains non-protein coding IPW-A exons flanking SNORD116 snoRNA genes cluster. Genomic deletion of PWScr is associated with PWS in human and growth retardation with 15% postnatal lethality in mice. In addition the PWS-locus contains another repeated snoRNA genes cluster - SNORD115. SNORD115 RNA was suggested to regulate alternative splicing and/or editing of serotonin receptor 5HT-2c pre-mRNA. The serotonin 5HT-2c receptor is a key contributor to obesity, autism and neurological diseases. Recently we generated a knock in (KI) mouse model containing a 5HPRT-LoxP-NeoR cassette (5LoxP) inserted upstream from the PWScr. When the cassette was inherited maternally in a paternally PWScr-deleted mouse model (PWScr (p-/m5LoxP)), growth retardation and postnatal lethality were rescued. We could demonstrate that the lack of expression of ncRNAs from the PWScr is primarily causative of PWS. However, an important question remained unanswered. Is the expression of both PWScr RNAs species or the lack of individual ncRNA causative of PWS in patients? Here, using transgenic mice we aim to answer this essential question and to investigate the impact of individual RNA species on growth phenotype. We will generate three different models expressing both PWScr RNAs species, or separate Snord116 RNA and IPW-A host exons, respectively, from the single controlled locus of transgene integration (ROSA26 locus). Detailed analyses of transgenic mouse lines in comparison with wild type and PWScr (p-/m+) animals will be performed to evaluate PWScr RNAs function. In addition, we would like to extend our study of PWScr (p-/m5LoxP) mice and investigate the function of another PWS locus encoded snoRNA - Snord115. The question whether Snord115 RNA involves in regulation of alternative splicing and/or editing of serotonin receptor 5HT2c pre-mRNA in vivo is still remains open. In wild type mice, expression of Snord115 RNAs was not identified in the choroid plexus, the brain area where serotonin 5HT2c receptor is highly expressed. We detected expression of Snord115 in the choroid plexus of PWScr (p-/m5LoxP) animals. Hence, PWScr (p-/m5LoxP) mouse model gives us a unique opportunity to investigate potential function of Snord115 RNA in regulation of 5Ht2cr pre-mRNA processing in vivo. Collectively, the proposed project will allow determining functional involvement of different ncRNA species in the PWS and in regulation of serotonin receptor activity. Determination of a molecular basis of PWS might lead to novel strategies for treatment.

• DFG Programme: Research Grants