Where and when does SNORD116 interact with its mRNA targets?

Funding Summary

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. 

Lay Abstract

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.

Research Outcomes: Public Summary

The smallest human causative deletions in patients with Prader-Willi Syndrome (PWS) delete a small region of chromosome 15 that contains the SNORD116@ locus (a group of 28 small nucleolar or snoRNAs), SNORD109a, and IPW genes. These are all non-coding RNAs, and thus there is still a lack of understanding about how deletions in SNORD116@ lead to the complex phenotypes of PWS. We previously showed that in mouse neuronal cell lines expressing Snord116, Nhlh2 mRNA, was protected from degradation, compared to cells without Snord116, and compared to cells with a mutant Nhlh2. These data suggested that Nhlh2 is a gene regulatory target of Snord116. The role of Nhlh2 as a transcription factor which regulates genes involved in the control of body weight, fertility and puberty are consistent with its role in protecting against the phenotypes seen in PWS. Additionally, other laboratories had shown that NHLH2 mRNA and protein were reduced in stem cells derived from PWS patients. However, we did not know where and when Nhlh2 and Snord116 interacted in brain tissue. Research from our funded study has now shown that Snhg14 -the host gene for Snord116, and Snord116 are co-expressed with Nhlh2 in neurons throughout the adult mouse brain, including Pomc neurons of the arcuate nucleus, which are an important mediator of energy balance signals for body weight and food intake regulation. Additional hypothalamus regions, including the suprachiasmatic nucleus (SCN), ventromedial nucleus (VMH), and paraventricular nucleus (PVN) also have both co-localized and non-co-localized neurons for Nhlh2 and Snhg14/Snord116, and we are examining the neuronal subtypes in these areas now.

In extending our analyses to other regions of the brain, we find that all areas of the brain examined in phase I of the project, including the habenula, hindbrain including the nucleus accumbens, cerebellum, and hippocampus show a significant correlation for co-expression in our analyses (P<0.001). In the habenula, we observe a distinct pattern of high expression for Nhlh2 in the medial, versus the lateral habenula, which corresponds to the co-localization patterns for Nhlh2 and Snhg14. In our study, 83% of medial habenular neurons express Nhlh2, while 52% express Snhg14. Of these neurons, there is a significant correlation for co-expression (P<0.0001). The medial habenula has been implicated in REM sleep, and inhibition of dopamine release in other areas of the brain, as well as roles in the susceptibility to nicotine addiction, avoidance response and male sexual behavior. In the hippocampus there are multiple co-localized cells in both the molecular, granular, and hilus layers of the dentate gyrus. The hippocampus is generally implicated in memory and learning, but newer studies have demonstrated that the mossy cells of the dentate gyrus are also glutamatergic and negatively regulate the GABAergic granular cells, together contributing to both cognitive and anxiety-like behaviors. Other brain areas are under intense investigation to identify the specific neuronal subtypes (where) and the patterns (when) of co-expression for Nhlh2 and Snhg14/Snord116.

In summary, we have made significant progress in understanding where and when Sngh14/Snord116 is colocalized with one of its mRNA target genes, Nhlh2. Based on the high magnification cell analysis, acquired 3D images, and probe location, we can conclude that the Snhg14 host transcript and Nhlh2 interact spatially. These data are helping us to form new hypotheses as to the role of Nhlh2 in the etiology of Prader-Willi syndrome, as well as providing general basic biology of where and when Snord116 expression prevents Nhlh2 mRNA degradation. With our collaborators in the Virginia Tech libraries, we have created an open-access repository of images and “stories” showing colocalization throughout specific neurons in the mouse brain. The viewer provides researchers, patients, and families the ability to view and zoom into images of mouse brain and examine co-expression as part of stories on regions. Human patient brain samples from control individuals and from patients with PWS, as well as a study on the mouse PWS model will be added to this repository in the future. The public repository, with is open access, free and interactive can be accessed at: https://digital.lib.vt.edu/collection/231c6f0d.

Research Outcomes: Publications

Analysis of SNHG14: A Long Non-Coding RNA Hosting SNORD116, Whose Loss Contributes to Prader–Willi Syndrome Etiology. Ariyanfar S, Good DJ. Genes 2023, 14(1), 97, https://doi.org/10.3390/genes14010097.