Engineering new molecular tools to study orphan small nucleolar RNAs in human cells

Recent advances in high-throughput sequencing have uncovered that the vast majority of the cellular genome encodes for a myriad of noncoding RNAs, which lack coding potential for proteins. Increasing evidence suggests that ncRNA classes are involved in important processes that help in decoding the genetic information within cells. A striking example is the family of small nucleolar RNAs (snoRNAs) that guide post-transcriptional modifications of key regulatory RNA´s which regulate essential cellular processes (e.g. transfer RNAs, ribosomal RNAs and small nuclear RNAs). Importantly, several novel snoRNAs without known target RNAs have been identified and these have been named “orphan snoRNAs”. Currently, there are 158 orphan snoRNAs in the human genome and a vast majority have no known function or target. Recent findings that orphan snoRNAs are dysregulated in human disease indicate that they may have evolved to accomplish important biological functions. One of these recent findings are the loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116, that contribute to Prader-Willi syndrome, the most common syndromic form of obesity in humans. Another study showed that the orphan snoRNA, U50, is transcriptionally downregulated in prostate cancer, and expression of U50 inhibits colony formation in prostate cancer cells. A third example, is orphan RNA SNORA42 which is frequently overexpressed in lung cancer and where high SNORA42 expression, show a significant correlation with poor survival. It is clear that there is a gap in the knowledge concerning the function of orphan snoRNAs. My project focused on developing molecular tools to study the biological role of snoRNAs, in human cells such as cancer- and stem cells. As a loss of function tool, this study optimized antisense oligos of a variety called locked nucleic acids (LNAs), that was able to specifically target an orphan snoRNA resulting in significantly decreased levels of expression. For gain of function studies, transient and stable transfection tools were created and optimized. A novel lentiviral system was engineered for stable overexpression in difficult-to-transfect cell lines using a candidate orphan called SNORD123 that our preliminary findings have shown to be significantly and differentially expressed during the process of stem cell differentiation towards all primitive embryonic germ layers (unpublished data). The lentiviral system was specifically tailored to mimic the human snoRNA biogenesis. Tools developed by this study will help shed new light into this fascinating and unexplored aspect of RNA biology and possibly provide novel mechanistic insights linking dysregulation of small ncRNA to human disease. Advisor: Roberto Munita, PhD (roberto.munita@med.lu.se) Master´s Degree Project, 30 credits in Molecular Biology (MOBM01) In collaboration with the Research School in Stem Cell Biology (VMFN25) Department of Biology, Lund University Department of Molecular Hematology, BMC, Lund University (Less)