The Snord116 gene is critical in PWS, but its normal function is incompletely understood. Dr. Good will establish an atlas of where and when the SNORD116 RNA is expressed in the developing mouse brain and how it interacts with one of its putative target genes, Nhlh2, to gain insight into the underlying molecular basis of PWS.
Dr. Theresa Strong, Director of Research Programs, shares details on this project in this short video clip.
The minimal deletion of chromosome 15q that still leads to Prader-Willi Syndrome (PWS) phenotypes includes just three expressed regions: SNORD116, SNORD109a, and IPW. However, even with this knowledge, there is still a need to understand why deletion of these non-coding RNAs lead to the complex phenotypes of PWS. This starts by identifying targets of the non-coding RNAs. Using a neuronal cell line, we recently demonstrated that Snord116 post-transcriptionally stabilizes Nhlh2 mRNA levels, increasing its half-life in the cell. Nhlh2 is a transcription factor that regulates downstream processes involved in body weight regulation, metabolism, and reproduction—all areas of physiology that are disrupted in a patient with PWS. However, no one has fully analyzed SNORD116 or NHLH2 co-expression in whole tissue sections, and a major unanswered question remains: Where and when is SNORD116 interacting with its target mRNAs in vivo?
We propose the testable hypothesis that Snord116 and Nhlh2 interact only at spatially and temporally distinct times (circadian, or adult/embryo) and places (neuron types, extra-neuronal tissues) that can be characterized using multiplex in situ hybridization. Multiplex in situ hybridization allows for up to 12 probes to be simultaneously visualized on a single slide. Sagittal and coronal sections through the brain and spinal cord, as well as muscle, pituitary, bone marrow and gonads will be examined using RNAScope and BaseScope multiplex in situ hybridization technology from ACD/Biotechne, to ask “where” Nhlh2, Snord116, and cell-specific type markers throughout the body are co-localized. Embryonic, as well as energy/metabolic signals in adult mouse tissues will allow us to ask “when” Snord116 and Nhlh2 are co-localized. We will compare results obtained using mouse tissues with human tissues (phenotypically normal, and from patients with PWS) from the NIH Brain and Tissue Banks to determine the answer to the critical question of whether mature, spliced NHLH2 mRNA is differentially regulated in brains and tissues from patients with PWS compared to normal controls. All images generated by these studies will be used to develop a free, online, interactive atlas of Snord116 expression, using Minerva authoring software, and hosted by the Virginia Tech Libraries Digital Repository, for open access use by PWS researchers and families.
We have the tools, reagents, genomic and genetic knowledge, and in situ hybridization expertise to attack the question of when and where NHLH2:SNORD116 are co-localized. The answers we seek will not only help to guide the PWS field on the function of SNORD116, but also contribute to the field of non-coding RNA biology and obesity/metabolism research by identifying new cellular expression for these two genes, and possibly, molecular targets of SNORD116 and NHLH2. These experiments are necessary to make actionable progress in Program 2a on the Research Goals for the Foundation for Prader-Willi Syndrome, genotype-to-phenotypes critical knowledge area.
Deborah Good, Ph.D.
Deborah Good, PhD