PORTFOLIO

Prader Willi Syndrome (PWS) is a rare congenital disease that results from the loss of expression of genes located on chromosome 15q11-13. Genes expressed from this locus are imprinted, meaning that only one of the two copies of the gene (maternal or paternal) is expressed. A number of studies implicate the loss of several paternally-expressed, non-protein- coding RNAs from this chromosomal region in the development of PWS, which manifests itself as excessive appetite and life threatening obesity along with mental and growth retardation, symptoms that are due to a hormonal imbalance. The only therapeutic approach that is available so far, i.e. administration of growth hormone, addresses only one phenotypic aspect of the syndrome. Interestingly, the noncoding RNAs that are encoded in the PWS locus are small nucleolar RNAs (snoRNAs) that are expressed predominantly in brain. Their function in the PWS is unknown, and thus understanding the molecular function of the snoRNAs implicated in PWS may be essential for developing new therapies.

Our project exploits recent developments in high throughput methods, like crosslinking and immunoprecipitation (CLIP) to study the functions of PWS associated snoRNAs and their processed variants.

snoRNAs are primarily involved in guiding modifications of ribose sugar moiety of ribosomal RNAs and small nuclear RNAs through RNA:RNA interactions. The snoRNAs that are encoded in the Prader Willi Syndrome locus do not bear complementarity to any conventional RNA targets and may therefore have entirely different functions. Previous studies attempted to relax the stringency of RNA-RNA complementarity in search of non-canonical targets, but this approach clearly yields a large number of false positives. Recently however, it has been shownthat these snoRNAs are processed into smaller, metabolically stable RNAs that could indeed have entirely different functions compared to the canonical snoRNAs. In this project, we use novel, genome-wide approaches to uncover the in-vivo RNA interactors of snoRNAs and their derived variants, with the hope that these will pave the way to new therapeutic strategies.

The project has been prematurely terminated. During the 6 months of project activity, we achieved the desired milestones that we set out for. In brief:

PAR-CLIP preformed on snoRNA core proteins in HEK293 cells confirmed the proof of principle. It successfully identified not only known snoRNAs but as well several other snoRNAs expressed in the HEK293 cell lines. As previously anticipated, novel sites of modifications in canonical targets of snoRNAs were identified and showed by primer extension assays. In addition, we identified several novel classes of targets that were not previously anticipated to be regulated by snoRNAs. Deep sequencing data on the small RNAs (15-30 and 20 to 200nt) obtained from total HEK293 RNA shows relevant processing of snoRNAs into smaller fractions from the 5’ and 3’ ends of the mature snoRNA.

PAR-CLIP experiments were subsequently optimized on the mice brain tissue and performed on microsections of freshly dissected mice brain revealed novel mouse brain specific snoRNAs while confirming the abundant expression of PWS linked snoRNAs. We completed a small RNA sequencing from total RNA obtained from mouse brain to identify the processing pattern of PWS linked snoRNAs. We identified that similar to snoRNAs derived from HEK293 cells, PWS associated snoRNAs are processed at their 3’ and 5’ ends and the processed variants might function differently. However, the small RNA derived from PWS loci are present in multiple copies and need to be further rigorously studied, as not only their abundance but as well their diversity is very large.