The role of variant (v)U1 snRNAs in development and disease.

Lead Research Organisation: University of Oxford
Department Name: Sir William Dunn Sch of Pathology

Abstract

The expression of the genes within our cells is strictly controlled according to the type of tissue and stage of development. For example, expression of genes whose products are needed at early stages of the development of embryos but not in adult cells will be switched on and off only at the right time. There are many mechanisms involved in regulating the switching on or off of gene expression and we have recently uncovered a new set of players in these processes-the variant U1 snRNPs. These novel complexes contain the nucleic acid, RNA together with proteins and there are indications that mis-regulation of the components of these complexes is associated with some neurodegenerative disease, including Spinal Muscular Atrophy (SMA) and some cancers, including Wilms' Tumour. Using the latest experimental techniques, we aim to fully characterise the composition of these complexes and identify which genes they regulate. This is a critical step towards understanding the exact roles these complexes play in human cells and how mis-regulation of the RNA or proteins could cause disease. The long-term goal of our studies is to develop therapies to combat the diseases associated with mis-regulation of these complexes.

Technical Summary

Our recent discovery of a large family of non-coding variant U1 small nuclear (sn) RNAs, (vU1s) which, like U1 snRNA (U1), can regulate recognition of splicing and polyadenylation signals in pre-mRNA, has uncovered new mechanisms controlling gene expression in human cells. vU1 genes are most highly-expressed in human embryonic stem cells (hESCs), in dedifferentiated cancer cell lines and the cells of patients with neurodegenerative disease, including Spinal Muscular Atrophy (SMA) and down-regulated upon differentiation. Thus, vU1s may play key roles in hESCs and early development and mis-regulation of these RNAs could cause disease.
We have shown that, like U1, one of the vU1s-vU1.8 is complexed with proteins in an snRNP. While some proteins are common to U1 and vU1.8 snRNPs, others are associated only with vU1.8, including DIS3L2, mutations in which cause Perlman Syndrome. The composition of snRNPs containing other vU1s has not been investigated. In addition, the direct pre-mRNA targets of any vU1 snRNP have not been identified.
The proposed research aims to further characterize the composition of the vU1.8 snRNP, which is found in HeLa cells and hESCs and the hESC-specific vU1.20 snRNP and to identify the direct targets of these RNPs using immunoprecipitation and RNA-seq. In addition, the role of DIS3L2 in the vU1 snRNPs will be investigated using immunoprecipitation, siRNA-mediated knockdown and analysis of DIS3L2 enzymatic activity. The role of vU1s in ESC maintenance and pluripotency will be investigated by knock down and over-expression of vU1s in hESCs. In addition, the expression of the full range of vU1s will be analysed by qRT-PCR in cells from patients with SMA and Parkinson's disease to determine whether mis-regulation of these RNAs is a common occurrence in neurological disease.
The proposed research will help to elucidate the roles vU1s play in human gene regulation and may yield insights into the molecular basis of human disease.

Planned Impact

The proposed research will benefit:
1) the scientists directly involved, as taking the research through to a successful conclusion will result in publications and training important for career advancement and the possibility of commercialization of discoveries. For example, Dr O'Reilly will add important skills in bioinformatics analysis of RNA-seq data to her portfolio of expertise and publish the results of the research, which will improve her chances of the next step to an independent scientific career. Dr O'Reilly and I will benefit by developing new expertise and knowledge investigating the nuclear role of DIS3L2, which will expand our scientific expertise. Dr O'Reilly and I will benefit from interacting closely with developmental biologists, neurobiologists and other molecular biologists with a primary interest in the regulation of gene expression at the level of pre-mRNA processing. In addition, Dr O'Reilly and I will benefit from interaction with the TDI, ISIS and MRCT, who will acquaint us with the pathways to commercialisation of basic research. This will enhance the possibility of commercialization of the proposed research or other research we are conducting and expand our expertise.
Dr Cowley will benefit from being involved in studying a new aspect of the control of stem cell maintenance and pluripotency, which dovetails with her own research. Dr Sareen will benefit from being involved for the first time in studying the structure and function of snRNPs and making important new contacts in Oxford.
Thus, the proposed research will underpin career advancement through the acquisition of new skills, new contacts and publications. In addition, the discoveries will benefit those scientists involved in taking them forward to commercialization.
2) scientists researching the control of gene expression at the level of pre-mRNA processing in normal and diseased cells, for example from SMA or cancer patients, as they can incorporate new discoveries into their own research within the next 2-5 years.
3) the private sector companies who could become involved in commercialization of discoveries (eg development of new SMA or anti-cancer treatments) within the next 10-15 years.
4) the medical professionals involved in treating neurological conditions or cancer if discoveries lead to the development of new treatments within the next 15-20 years.
5) the NHS, if the discoveries lead to therapies for neurological conditions or cancer, within the next 15-20 years.
6) the general public, if treatments are developed for currently untreatable conditions, within the next 15-20 years.

Thus, the research has the potential to contribute to the health of the nation by treatment of neurological conditions and cancer and to contribute to the wealth of the nation through the development and worldwide sale of new drugs.
In addition, the proposed research will provide new UK expertise in the study of the control of early developmental decisions and will strengthen the UK's research effort into SMA and cancers associated with mis-regulation of DIS3L2 (eg in Perlman Syndrome).
 
Description 2015 Goodger and Schorstein Scholarship
Amount £2,000 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 11/2015 
End 11/2015
 
Description Oxford Cancer Research Centre Development Fund
Amount £10,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 03/2018
 
Description The human pol II-transcribed snRNA genes; a model for gene-type specific transcription.
Amount £468,091 (GBP)
Funding ID BB/R016836/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 06/2021
 
Description Wellcome Trust Project Grant (Connecting transcription of small nuclear RNA genes with RNA 3 end formation)
Amount £244,168 (GBP)
Funding ID 081312/Z/06/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2007 
End 12/2009
 
Description Wellcome Trust Project Grant (The role of the pol II CTD in expression of human snRNA genes.)
Amount £396,633 (GBP)
Funding ID 088542 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2009 
End 07/2012
 
Description Wellcome Trust Senior Investigator
Amount £750,000 (GBP)
Funding ID 106134/Z/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2018
 
Description Expression of human snRNA genes 
Organisation University of Toulouse
Country France 
Sector Academic/University 
PI Contribution Transcriptional analysis and training of techniques and bioinformatic analysis was carried out in my lab.
Collaborator Contribution Our partner has made new cell lines where factors involved in snRNA gene expression have been knocked out.
Impact We are currently writing a paper on the research outcomes.
Start Year 2018
 
Description Neurons 
Organisation Cedars-Sinai Medical Center
Country United States 
Sector Hospitals 
PI Contribution All the experimental analysis on the RNA from neural cells from unaffected or disease individuals.
Collaborator Contribution They derived the neural cells from fibroblasts from unaffected or disease individuals.
Impact Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease. Vazquez-Arango P, Vowles J, Browne C, Hartfield E, Fernandes HJ, Mandefro B, Sareen D, James W, Wade-Martins R, Cowley SA, Murphy S, O'Reilly D. Nucleic Acids Res. 2016 Dec 15;44(22):10960-10973.
Start Year 2014
 
Description Parkinson's disease 
Organisation University of Oxford
Department Department of Computer Science
Country United Kingdom 
Sector Academic/University 
PI Contribution We carried out all the experimental work with RNA from neurons from unaffected individuals or individuals with Parkinson's disease.
Collaborator Contribution They derived neurons from fibroblasts from unaffected or affected individuals.
Impact Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease. Vazquez-Arango P, Vowles J, Browne C, Hartfield E, Fernandes HJ, Mandefro B, Sareen D, James W, Wade-Martins R, Cowley SA, Murphy S, O'Reilly D. Nucleic Acids Res. 2016 Dec 15;44(22):10960-10973.
Start Year 2014
 
Description Stem cells 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution All of the experimental work with stem cells and RNA from stem cells.
Collaborator Contribution Provided stem cells for experiments.
Impact Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease. Vazquez-Arango P, Vowles J, Browne C, Hartfield E, Fernandes HJ, Mandefro B, Sareen D, James W, Wade-Martins R, Cowley SA, Murphy S, O'Reilly D. Nucleic Acids Res. 2016 Dec 15;44(22):10960-10973.
Start Year 2014
 
Description Splicing conference, Bermuda 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk at a specialist conference on splicing
Year(s) Of Engagement Activity 2017