Dr. Grzechnik’s lab is interested in uncovering the biological mechanisms underlying PWS. The deletion in the PWS locus affects the regulation of gene expression in neurons, but scientists are not exactly sure how this mechanism works. This current project is testing how coding and non-coding regions of the human genome are transcribed in cells lacking specific PWS-related non-coding RNAs, which will help to identify regulators of gene expression in PWS.
Dr. Theresa Strong, Director of Research Programs, shares details on this project in this short video clip.
This grant was funded in part by FPWR-UK.
This project aims to uncover the biological mechanisms underlying Prader-Willi syndrome (PWS) which is the most common syndromic cause of life-threatening obesity in humans. My goal is to pinpoint genetic elements missing in PWS that are required for normal functioning of neuronal cells. Development and maintenance of neurons are dictated by a specific gene expression pattern where information encoded in DNA is transcribed to coding mRNAs which are used as templates for protein synthesis. This simple chain is controlled by a sophisticated network of regulatory factors, proteins and non-coding RNAs (ncRNAs). The deletion in locus q11-13 of chromosome 15 (PWS locus) affects the regulation of gene expression in neurons and manifests as PWS, but the exact mechanism is not known. The deleted fragment harbours small nucleolar RNAs (snoRNAs) which have been assumed as major players in neuronal development. However, despite extensive studies, there is no clear indication of how these snoRNAs may regulate gene expression. Recent research uncovered that PWS-associated snoRNAs can be also included into two different long non-coding RNAs (lncRNAs) and form so-called sno-lncRNAs or SPA-lncRNAs. Owing to snoRNAs secondary structures and association with proteins, such snoRNA-lncRNA hybrids are protected from degradation. These lncRNAs have the potential to sequester factors that control mRNA synthesis and therefore, gene expression.
Dr. Grzechnik will investigate roles in RNA transcription for particular ncRNA classes transcribed from the PWS locus. For the first time in PWS research, I will test how coding and non-coding regions of the human genome are transcribed in cells lacking specific PWS-related ncRNAs.
Understanding ncRNA functions in PWS will prove fundamental for future therapeutic approaches. Here, I will identify PWS-associated bona fide regulators of gene expression. This will allow the identification of the defective cellular pathways that are the primary source for the global deregulation of gene expression that triggers PWS.
This project can be developed further. Identification of the regulatory ncRNAs will be the first step to understand their functions. Additional experiments may uncover various roles of these ncRNAs during different stage of neurodevelopment. Also, experimental work is needed to assess how to rescue defective transcriptional pathways in affected cells.
The outcomes of this project will not result in the immediate therapeutic approach. However, they will be absolutely essential to lay a foundation for future gene therapies and will have a clinical effect within the next 10-15 years. The impact of this research can be described by paraphrasing Edward Teller’s quote – “The science of today is the medicine of tomorrow”.
Research Outcomes: Public Summary
Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders such as Prader-Willi syndrome (PWS) which results from the deletion of an imprinted locus on chromosome 15. We analysed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5’ snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS.
We show that the lack of these ncRNAs decreased transcription of neurodevelopmental genes and increased transcription of factors that negatively affect cellular growth and mediate apoptosis. The region downstream of the SNURF-SNRPN gene, SNHG14, that encompasses the minimal deletion resulting in PWS, produces one of the most abundant chromatin-associated RNAs in iPSCs. The chromatin association of PWS ncRNAs points towards their possible function in transcription regulation. SPA- and sno-lncRNAs display unique structural properties: the snoRNA structures at 5’ or both ends make them relatively resistant to prevalent exonucleolytic activities. Thus the intervening sequence can be used as a sponge RNA to sequester transcription and splicing factors and hence affect gene expression. Our analysis of chromatin-associated RNA, that can be used as a proxy for active transcription of protein-coding genes, uncovered that SPA- and sno-lncRNAs control the transcription of many genes that regulate neurodevelopment including NRXN1, NLGN1, and FAT3. We found that SPA- and sno-lncRNAs depletion decreased transcription levels for many genes that contribute to the formation of neuron specific structures – axons and synapses, as well as genes required for proper cell adhesion and cell-cell signalling. Interestingly, a significant population of genes which were upregulated by SPA- and sno-lncRNAs knockdown, are involved in the negative regulation of cellular metabolism and apoptosis. One possible explanation of this phenotype is that misregulation of neurodevelopmental genes that may result in abnormal differentiation is countered by repression of cell growth and ultimately, cell death. The global effect of SPA- and sno-lncRNAs depletion on RNA levels was completely lost in the total RNA fraction, representing mainly steady-state cytoplasmic RNAs. This is consistent with the observation in a mouse model where only seven genes including transcription factor Mafa and growth suppressor Necdin were upregulated by deletions in PWS locus. In human cells, deregulation of transcription caused by the absence of SPA- and sno-lncRNAs may feature in total RNA levels in later stages of neurodevelopment, when the expression of these genes is essential to support neuronal maturation. Moreover, adjusting mRNA to optimal concentration in dynamically differentiating stem cells, when the gene transcription is affected, may not be as responsive and efficient as in healthy cells. Thus, it may introduce errors in gene expression that accumulate during development and, as a consequence, manifest as PWS. Such a pathological pattern, where a transcriptional regulation imbalance emerges already in stem cells, impacts neuronal development and results in a late onset disease, has been reported for many neurodevelopmental disorders. Mild but persistent effects on overall gene expression may be the reason why the deletions in the PWS locus are not lethal. However, as neurons progress through highly organised processes of differentiation, migration and functional activation, any disruption in gene expression may affect the development of human brain, leading to the manifestation of neurodevelopmental disorders.
Overall, we demonstrated for the first time, a global decrease in the transcription of neurodevelopmental genes caused by the lack of transcripts from the 15q11-13 locus. Thus, we concluded that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature important for neuronal differentiation and development. Moreover, our results emphasize the importance of studying the direct effect on transcription in PWS models and provided an easy-to-employ alternative to genomic deletions in PWS studies.
Research Outcomes: Publications
Non-coding RNAs Associated with Prader-Willi Syndrome Regulate Transcription of Neurodevelopmental Genes in Human Induced Pluripotent Stem Cells. Sledziowska M, Winczura K, Jones M, Almaghrabi R, Mischo H, Hebenstreit D, Garcia P, Grzechnik P. Human Molecular Genetics (2022): doi: 10.1093/hmg/ddac228
Pawel Grzechnik, PhD
University of Birmingham