The vast majority of RNA molecules are non-coding and have different but important functions. Among these are snoRNAs, which are small non-coding RNAs that accumulate in the nucleus and direct post-transcriptional modifications of many other RNA molecules. Based on the characteristic conserved sequences, snoRNAs are divided into two subgroups, SNORD and SNORA. In this thesis, we focused on SNORD molecules or C/D box snoRNAs. The majority of these RNAs guide 2' O methylation of ribosomal RNA, but there are also those that have unknown functions or targets, and they are referred to as snoRNA orphans. The group includes SNORD116 gene cluster, which is defined as the minimal critical region for Prader-Willi syndrome (PWS). PWS is a rare genetic neurodevelopmental disorder caused by a paternal deletion of genes on chromosome 15 and can lead to life-threatening obesity, among other symptoms. Since the SNORD116 gene cluster is absent in all PWS patients, it is considered a key genetic factor in the syndrome’s development.
In this master's thesis, we optimized the COMRADES method protocol to identify RNA targets of SNORD116. The method is based on in vivo cross-linking of RNA duplexes with psoralen-TEG azide, forming covalent bonds between two RNA strands upon long wavelength UV irradiation. This process is followed by the isolation of total RNA from cells, partial fragmentation, and biotinylation of cross-linked duplexes, which are then affinity captured using a "pull down" assay. Cross-linked fragments are separated by polyacrylamide gel electrophoresis, after which we ligate the ends of both cross-linked molecules and use short-wavelength UV light to break the covalent bonds between the RNA strand pair. This results in hybrid single-stranded RNA molecules from which we synthesize a cDNA library and attempt to identify pairs of SNORD116 and its target RNAs through sequencing and bioinformatic analysis.
We successfully optimized several protocol steps and added a step to enrich the selected RNA. By hybridizing with biotinylated DNA probes and using affinity capture techniques, we increase the relative amount of SNORD116 in the sample and avoid significant losses. However, due to the extensive experimental work in this thesis, we were not able to identify the RNA targets of SNORD116 in the end. Nevertheless, the optimized protocol will serve as a foundation for future experiments aiming to uncover the functions of this enigmatic gene cluster.
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