We expect that discovering the direct functions of snoRNAs will uncover new mechanisms – as well as revealing the fundamental basis of PWS. We propose to create a wide picture of RNA-RNA and RNA-protein interactions during the development of brain cells, focusing on interactions of SNORD116, as well as SNORD115 and other ncRNAs synthesized from SNHG14. We will identify their direct interacting RNA and protein partners. Using mutant cell lines we will find specific changes in other RNAs and proteins resulting from their absence during neuronal differentiation. During the current project, we expect to make important discoveries about how brain-specific snoRNAs, particularly SNORD116, are regulated and function.
Dr. Theresa Strong, Director of Research Programs, shares details on this project in this short video clip.
Lay Abstract
The genetic information in all organisms, including humans, is encoded in the sequences of very long molecules of DNA. However, to be used the data must be retrieved by copying into a related molecule called RNA. In this medium the information can be used to control all cellular processes. In consequence, RNA metabolism lies at the heart of the information processing systems that fundamentally distinguish living organisms from sets of biochemical reactions. The mRNAs encode the information needed to direct the synthesis of proteins. Others, termed non-coding RNAs (ncRNAs), function more directly. Characterized ncRNAs play many different roles, particularly in the machinery of protein synthesis, but it is very likely that more functions remain to be found. Prader-Willi syndrome can be caused by loss of ncRNAs that are synthesized from the SNHG14 locus. Among these are two sets of related RNAs collectively called SNORD115 and SNORD116, which are made by being cut out of a very long SNHG14 ncRNA. A gene deletion that removes only the part of SNHG14 that generates SNORD116 is enough to cause PWS. So these RNAs clearly have important functions - but we do not know what they are. SNORD116 is part of a much larger family of RNAs, termed small nucleolar RNAs (snoRNAs), that our group has studied since the 1980s. Most snoRNAs are expressed in all cells and are important in the manufacture of ribosomes, tiny machines that synthesize all the proteins. However, SNORD116 (and SNORD115) seem to be different. They are mainly expressed in the brain and do not seem to function in ribosome synthesis. We expect that discovering the direct functions of these snoRNAs will uncover new mechanisms – as well as revealing the fundamental basis of PWS. Many diseases start with small defects in the genome that ultimately cause serious health problems; as a small engine defect will ultimately cause a vehicle breakdown. Understanding of the first steps and the actual function of the defective component are needed for early interventions. In this project we will use methods well established in our lab to identify those early steps. We propose to create a wide picture of RNA-RNA and RNA-protein interactions during the development of brain cells, focusing on interactions of SNORD116, as well as SNORD115 and other ncRNAs synthesized from SNHG14. We will identify their direct interacting RNA and protein partners. Using mutant cell line we will find specific changes in other RNAs and proteins resulting from their absence during neuronal differentiation. We will then test a set of specific hypotheses for the molecular basis of these changes. The applicants are RNA-biologists, who bring insights and techniques to understand changes in neuronal RNA metabolism that ultimately lead to PWS. During the current project, we expect to make important discoveries about how brain-specific snoRNAs, particularly SNORD116, are regulated and function. Defects in these processes ultimately cause PWS and, in subsequent work, we will collaborate with neurobiologists to apply this basic understanding towards the development of molecular treatments, e.g. based on RNA therapeutics.
Clinical diagnosis of PWS will occur months or years after the differentiation defects that we characterize. By that time, the primary causes will likely be hard to identify. This is also the case for many other impairments that start early during embryonal development. We therefore analyzed developmental time courses, with the aim of characterizing the initial defects that arise from the absence of SNORD116.
The small nucleolar RNAs SNORD115 and SNORD116 are expressed from tandem arrays of genes located within the very large non-coding RNA SNHG14. The project to date, has identified molecular alterations during neurodevelopment in the absence of transcription from specific regions SNHG14 that include the snoRNA gene clusters. These indicate the pathways affected and may provide targets for therapeutics. Since, PWS presentation changes during development, with defined phases, the possibility for postnatal intervention remains open.
In a neuronal model, we tested mechanisms by which RNAs encoded by SNORD115 and SNORD116 clusters might alter gene expression. Partially overlapping changes in RNA abundance and predicted translation were observed on loss of either snoRNA. It can be envisaged that the effects of the SNORD115 and SNORD116 might be mediated by multiple mechanisms. Common phenotypes could reflect effects on chromatin organization in and around SNHG14, or “mass-action” effects of the snoRNAs, e.g. on RNA-protein condensates. However, micro-deletion of SNORD116 but not SNORD115 has been reported to result in PWS. We therefore focused on changes specifically shown by the cell line lacking SNORD116 expression, relative to cells lacking SNORD115 and the parental line.
This generated a much smaller list of genes with significantly changed expression. Most mRNAs that were specifically altered have not been extensively studied, but available publications reveal neuronal-enriched expression and strong links to PWS-relevant phenotypes. A small number of non-coding RNAs were also found to be altered in expression. Strikingly, 3 out of 4 were transcribed antisense to the promoter regions of mRNAs that also showed altered expression. The molecular basis for this remarkable observation will now be determined.
Particularly striking changes were seen for a small subset of Protocadherin (PCDH) mRNAs. This large family of cell-adhesion proteins play key roles in intercellular signaling, with clustered genes predominately showing neuronal expression. No clear changes were seen for non-clustered PCDH genes, whereas the PCDH-alpha and PCDH-gamma clusters showed distinctly different expression patterns in H116 relative to the WT or H115 cells. These may be significant for PWS, since both PCDH-alpha and PCDH-gamma have previously been implicated in neurodevelopmental disease. Future work will assess whether the PCDH genes are direct targets for SNORD116 and potential mechanistic links.