Ultra-structural and dynamic analyses of the sub-cellular organisation of messenger RNA biology
Lead Research Organisation:
University of St Andrews
Department Name: Biology
Abstract
Messenger RNA is crucial to cellular gene expression because it interprets genetic information encoded by DNA. The mechanisms involved include two major events of mRNA production and use. Firstly, splicing is particularly important because it creates mature mRNA molecules and allows flexibility of the genome by creating different splicing isoforms. Secondly, sub-cellular transport of mRNA has been shown to be vital to embryonic development in species such as Drosophila, as well as also regulating pluripotency of mouse embryonic stem cells.
Henceforth, the physical organisation of mRNA splicing within the nucleus of a cell has not been fully understood. Hence, this project aims to gain an insight into some of the structures involved in alternative splicing and mRNA trafficking. Cells from the eye lens will be used because they present an effective model of cellular development. This is because, as cells in the lens persist throughout the life span of an organism, the model provides an entire developmental history to examine. Furthermore, structures within the nucleus specific to RNA processing, show distinct patterns of alteration during lens development.
MBNL1 and CELF1 proteins are alternative splicing factors that have antagonistic roles in controlling the developmental switch between foetal and adult isoforms. Furthermore, MBNL1 has been shown to co-localise with mRNA in the cytoplasm and regulate its localisation. Therefore, these structures will be mostly the focus of this investigation. Previously, the protein structures have been tagged with GFP and the recently innovative molecule: mini Singlet Oxygen Generator (miniSOG). This has allowed one to observe the localisation of these structures within the nucleus and cytoplasm. Now, the aim is to examine their sub-cellular dynamics in lens epithelial cells, intact eye lenses and differentiated muscle cell lines. This will allow one to analyse the antagonistic effects of MBNL1 and CELF1 within the splice-site and mRNA localisation in the cytoplasm. Techniques to be used will include those in Microscopy (particularly Fluorescence, Electron, Live Cell Time Lapse, Photobleaching and FRET), Molecular Biology, Biochemistry and Mass spectrometry.
Henceforth, the physical organisation of mRNA splicing within the nucleus of a cell has not been fully understood. Hence, this project aims to gain an insight into some of the structures involved in alternative splicing and mRNA trafficking. Cells from the eye lens will be used because they present an effective model of cellular development. This is because, as cells in the lens persist throughout the life span of an organism, the model provides an entire developmental history to examine. Furthermore, structures within the nucleus specific to RNA processing, show distinct patterns of alteration during lens development.
MBNL1 and CELF1 proteins are alternative splicing factors that have antagonistic roles in controlling the developmental switch between foetal and adult isoforms. Furthermore, MBNL1 has been shown to co-localise with mRNA in the cytoplasm and regulate its localisation. Therefore, these structures will be mostly the focus of this investigation. Previously, the protein structures have been tagged with GFP and the recently innovative molecule: mini Singlet Oxygen Generator (miniSOG). This has allowed one to observe the localisation of these structures within the nucleus and cytoplasm. Now, the aim is to examine their sub-cellular dynamics in lens epithelial cells, intact eye lenses and differentiated muscle cell lines. This will allow one to analyse the antagonistic effects of MBNL1 and CELF1 within the splice-site and mRNA localisation in the cytoplasm. Techniques to be used will include those in Microscopy (particularly Fluorescence, Electron, Live Cell Time Lapse, Photobleaching and FRET), Molecular Biology, Biochemistry and Mass spectrometry.
Organisations
People |
ORCID iD |
| Judith Sleeman (Primary Supervisor) |
Publications
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M010996/1 | 01/10/2015 | 31/03/2024 | |||
| 1645195 | Studentship | BB/M010996/1 | 01/10/2015 | 31/08/2020 |
| Description | We look into how a multi-systemic disease called Myotonic Dystrophy Type 1 affects the health of cells in the eye lens. Normally, cells in the eye lens have to be quite robust in handling stressful situations, because they are so exposed to the external environment. What we have found is that cells in disease aren't handling stress as efficiently, and this might be impacting their maintenance. This is important because cataract is the most abundant symptom in this condition. Thus, it's important to speculate if this inefficiency is contributing to cataract. |
| Exploitation Route | We have identified an impairment in stress regulation within Myotonic Dystrophy Type 1. The next step now is to see if this impairment is contributing to the symptoms seen in the condition, thus taking the cellular change that we have observed and seeing how it impacts the tissue. The eye lens is composed of different kinds of cells, primarily composed of the epithelial layer and the fiber cells. The observations were made on the epithelial layer and thus it's now important to see what is happening in the fiber cells. We have also been able to successfully produce hydrogels for our cells to mimic the lens environment, which will be important for the next researcher to use to continue this research into how the lens as a whole is impacted in disease. Moreover we have developed techniques in electron microscopy where we can look at the structures we have investigated in a lot more detail. |
| Sectors | Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Honor Fell/Company of Biologists Travel Awards |
| Amount | £500 (GBP) |
| Organisation | British Society for Cell Biology |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 08/2016 |
| End | 08/2016 |
| Description | ISSF |
| Amount | £625 (GBP) |
| Organisation | Wellcome Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 11/2017 |
| End | 12/2017 |
| Description | MDUK Project Grant: Involvement of the paraspeckle nuclear domain in Myotonic Dystrophy Type 1 |
| Amount | £174,803 (GBP) |
| Organisation | Muscular Dystrophy UK |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 09/2016 |
| End | 04/2021 |
| Title | Volocity |
| Description | Volocity is an imaging analysis software that uses algorithms in its protocols to analyse the contents of microscopy images. I used this a lot in my PhD where I designed protocols to find and track different objects in my images and videos and analyse them based on size, content, speed, etc. I also used it to analyse correlation between the objects in the protocols I designed. |
| Type Of Material | Data analysis technique |
| Provided To Others? | Yes |
| Impact | Without it, my correlation analyses between images wouldn't have been possible. I would have been able to track objects by size and count, but it wouldn't be as accurate as the software provides. This is particularly notable when measuring the volume of objects in 3D images |
| Title | Website Development for IPF stakeholders in MCN NHS Fife sector |
| Description | NHS Fife contain all diseases information and advice on their website for people and physicians to use. The section on Idiopathic Pulmonary Fibrosis was lacking in information and was deemed not very useful to any stakeholder. It was particularly difficult because as a disease, it's quite unknown and largely uncharacterised. In my internship I improved the contents on the MCN website for IPF, including different domains about the illness, and where to seek advice in Fife. This was to help everyone access all the information they would need regarding the illness. The MCN is mainly for physicians, but during my internship they opened the website to the public. |
| Type | Health and Social Care Services |
| Current Stage Of Development | Early clinical assessment |
| Year Development Stage Completed | 2017 |
| Development Status | Under active development/distribution |
| Impact | After interviewing different IPF patients and different respiratory physicians, it was clear that they wanted a clearer resource into the knowledge of this disease, and its associated care. They agreed that improving the website achieved this. |
| Description | British Science Week (Workshop) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | As part of British Science week in partnership with the University of Aberdeen, a group of postgraduate students participated in "Science Speed dating" with the general public. This involved every researcher giving a short 2 minutes talk about their research to members of the general public, and they in turn had 2 minutes to ask any questions that they had about your research. |
| Year(s) Of Engagement Activity | 2016,2018,2020 |
| Description | First Chances Programme (School visit) |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Schools |
| Results and Impact | First chances is a programme for high school students to come to the university and try different techniques/workshops in sciences, medicine and mathematical departments. I assisted the medical school in teaching them about human bone structure and content, and how balance and proprioception is achieved. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Restart the Heart day (general public CPR workshop) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Restart the heart was a day to teach the general public how to do CPR and use a defibrillator. This was to raise awareness on how you could save a life in an emergency. It involved us standing in the street with human-like dummies and recruit people on the street to teach and have a go at CPR and using the defibrillator. |
| Year(s) Of Engagement Activity | 2018 |