Remodelling of cellular RNA Metabolism during Prader-Willi syndrome

The major aim of this project is to determine how cellular RNA metabolism is remodelled during inherited genetic disorders, specifically using Prader-Willi syndrome (PWS) as a model. PWS is a neurodevelopmental disorder characterised by early onset of hyperphagia and occurs in ~1 in 10-30,000 people. This disorder is caused by gene expression changes in the hypothalamus, a small and central region of the brain involved in metabolic homeostasis, which involves the regulation of hunger. PWS is typically caused by loss of an imprinted genomic region that includes multiple, tandem copies of two small nucleolar RNAs (snoRNAs), snoRD115 and snoRD116. These are excised from the Small Nucleolar RNA Host Gene 14 (SNHG14). The smallest known deletions generating PWS remove only the snoRD116 repeats. Most snoRNAs are expressed in all cell types and are integral to ribosome synthesis. However, both snoRD115 and snoRD116 appear not to be involved in ribosome synthesis and are instead known as 'orphan' snoRNAs, since there aren't any known direct targets for either. Throughout this project, the Lund human mesencephalic (LUHMES) cell line will be employed. These are human embryonic neuronal precursor cells that can be differentiated into functional, dopaminergic neurons. To determine the RNA metabolism underlying PWS, this project will currently focus on two main questions.

First, the question of why snoRNAs are neuronal specific will be tested.
Initial RNAseq data indicated that changes in transcription or splicing of the host gene aren't responsible for snoRD116 accumulation. This data will be analysed further to confirm these conclusions. We will also test the hypothesis that stabilisation of snoRNAs is due to the neuronal expression of Fibrillarin (FBL) homolog, Fibrillarin-like 1 (FBLL1). FBL is one of the four core proteins that snoRDs associate with to form small nucleolar ribonucleoproteins (snoRNPs). However, preliminary analyses showed that FBL expression is lost during neuronal differentiation, whereas FBLL1 is strongly upregulated. This suggested that snoRD116 accumulation during differentiation may require stabilisation by FBLL1. Binding of snoRD115/116 to core proteins NOP58 and FBL will be determined. In contrast, RNAseq data for snoRD115 indicates the appearance of specific transcripts during differentiation, so the endpoints and timing of these transcripts will be determined.

Secondly, the question of why snoRD115 and snoRD116 loss alters the abundance of many ncRNAs and mRNAs in late neurodevelopment will be answered by determining the primary targets and mechanisms of snoRNAs.
Several tests will be performed to determine this. Small RNA sequencing will be performed to confirm the relative expression of different isoforms of snoRD115, snoRD116 and other neuronal-specific snoRNAs. Informatics will be used to test potential sequence complementarity to target mRNAs for the most expressed snoRNA sequences. RNA from cells lacking snoRD116 will also be analysed for splicing defects and potential target introns tested for complementarity. Moreover, whether ectopic expression of snoRD115 and snoRD116 suppress changes in gene expression in deletion mutants will be determined, which would potentially reveal which effects are caused by the loss of mature snoRNAs. Finally, RNA-RNA interactions will be determined using proximity ligation techniques that are either protein-based, such as CLASH, using tagged proteins, or hiCLIP, using antibodies, or RNA-based, such as COMRADES, using oligonucleotide selection.

With execution of this research on PWS, insights could be applied to other neuronal snoRNAs and potential targeted interventions for RNA-linked cognitive-impairment disorders could be consequently developed. Furthermore, the early diagnosis and facilitation of future, pre-emptive treatments could also be potentially improved.