Prader-Willi syndrome (PWS) traits are genetically determined by the loss of expression of genes from the paternally inherited small region located on chromosome 15. These genes are transcribed to brain-specific small nucleolar RNAs (snoRNAs) whose function is still unknown. snoRNAs were traditionally assigned a role in ribosomal RNA processing and modification through base-pairing with target RNAs, yet PWS snoRNAs display no sequence complementarity to canonical targets. This suggests that PWS snoRNAs might target other RNA species, thereby exerting alternative functions. Notably, a number of snoRNAs with intriguing functions were recently described, but little is known about their mechanism of action. Identifying targets of PWS snoRNAs would provide insight into pathological mechanisms underlying PWS at molecular level. In a pilot study backed by FPWR, we compared several experimental protocols, based on cross-linking RNAs in live cells followed by high-throughput sequencing of hybrid pairs, for their feasibility to interrogate RNA-RNA interactions genome-wide. We were able to capture validated RNA-RNA interactions formed by highly expressed RNA species, whereas PWS snoRNA-containing reads were scarce, precluding identification of RNA targets with high reliablity. Here, we propose to introduce modifications to our platform and evaluate the improved experimental protocol with regards to increased sensitivity for detection of RNA-RNA interactions. The optimized approach will be applied to unbiasedly screen for RNA targets in cells that express PWS snoRNAs. After the RNA targets are identified, we will monitor the effects that PWS snoRNAs exert on them. We will analyze potential changes in expression levels, splicing profile and/or nucleotide modifications of targeted RNAs in cells that do not endogenously express PWS snoRNAs before and after a specific snoRNA is introduced in them.
Dr. Theresa Strong describes this grant, why we are excited about it and what the long term contributions of this project may be in our Research Grants Program Update Webinar, Spring 2018. You can learn more about this specific project in this video segment.
Research Outcomes: Public Summary
A deletion within a paternally expressed locus on the proximal long arm of chromosome 15 (15q11-13), harboring a number of small nucleolar RNAs (snoRNAs; SNORD115, -116, -109A/B, -108, -107, and -64) is considered to be the primary genetic determinant of PWS. Recent case studies have pinpointed lack of SNORD116 expression as the minimal requirement underlying the PWS phenotype. PWS locus snoRNAs are brain-specific and their RNA interacting partners are unknown. Using an experimental approach that captures transient RNA:RNA interactions in live cells and allows for their unbiased detection, we have identified several potential targets of SNORD116, most of which belong to mRNAs. The hits, however, need to be confirmed in validation experiments. Once validated, RNA targets will be the focus of mechanistic studies with the ultimate goal of revealing PWS pathological processes at molecular level.
Research Outcomes: Publications
Neuronal differentiation induces SNORD115 expression and is accompanied by post-transcriptional changes of serotonin receptor 2c mRNA. Bratkovič T, Modic M, Camargo Ortega G, Drukker M, Rogelj B . Scientific Reports. 2018 Mar 23;8(1):5101.
Functional diversity of small nucleolar RNAs. Bratkovic T, Božic J, Rogelj B. Nucleic Acids Res 2020; 48(4): 1627-1651.
Tomaž Bratkovic, PhD
University of Ljubljana, Slovenia