How do RNA-binding proteins control splice site selection?

Lead Research Organisation: University of Leicester
Department Name: Molecular and Cell Biology

Abstract

Most genes contribute to the life of an organism by encoding proteins. The level of protein expressed depends on the level of transcription (in which an RNA copy of the gene is made) and the level of translation (when the RNA copy is used as a template for protein synthesis). In animals and plants, there is a step in between in which most of the RNA is spliced out of the original RNA copy, leaving a much smaller RNA sequence to be translated. In complex organisms, particularly vertebrates, the RNA copies of many genes can be spliced in a number of different ways. This means that a number of different proteins can be produced from one gene. This is amazing, and it happens to the greatest extent in the human brain. Unlike the other processes, splicing does not so much control the LEVEL of protein made, but rather it determines WHICH protein is made. We can squeeze around 8-10-fold more proteins out of our genes than would have been expected, and the different variants are expressed in different parts of the body, different cell types, at different stages in the life of a cell, or in response to ageing or disease.

This incredible extra layer of flexibility has been achieved by weakening the simple system for recognising splice sites that is seen in lower eukaryotes (like yeast). Instead, our genes are full of sequences that could be splice sites. How does the cell recognise the right sites? How does it switch when required from one set of sites to another? These processes are controlled by a large number of proteins that bind to the RNA and activate or repress potential splice sites. How do they do this? This is a really critical process, essential for life, required for memory, diurnal rhythm, development and almost every other healthy biological process, a cause and contributor to disease when it goes wrong, a potential target for therapies... but the answer to the last question is that we do not know, despite years of investigations.

Splicing is complex. There have recently been stunning advances in understanding the process of splicing the RNA after the right sites have been identified, but our understanding of how the sites are identified has barely changed in 25 years. Our conceptual principles have been outstripped by data. One of the most unsettling things we have come to realise is that some portions of the RNA can be bound by numerous proteins, activators and repressors, all of which have effects on the outcome, but they cannot all fit on at once. Do the proteins bind independently, weakly and transiently, and the outcome is a matter of chance that a particular protein is bound at a particular moment, do they bind stably in defined combinations, where several combinations might somehow permit splicing and others block it, or do activators and repressors actually bind competitively at a single crucial site? How does the next step work? How do activators activate or repressors repress? Do they form direct contacts with spliceosomal proteins or alter the flexibility and freedom of movement of the RNA or each other? How do they contact each other?

We can answer these questions. We have developed a way of looking at single molecules of RNA in nuclear extracts (which support splicing). By labelling just two types of protein with fluorescent dyes, we can determine whether they both bind the same molecule of RNA and how many of them are bound. We have also tested whether a particular activator protein communicates with splice sites by 3D diffusion or by propagating complexes along the RNA. Based on such experiments, we have recently published a breakthrough paper that describes evidence for new molecular mechanisms for the activator. By testing lots of proteins in pairs, we can determine their binding patterns, and then their modes of communication. We propose to use innovative methods to look at the properties and interactions of the proteins, and then observe their binding in real time.

Technical Summary

The purpose of this research is to understand the molecular mechanisms by which RNA-binding proteins determine whether a particular pre-mRNA exon or splice site is spliced. Exons and nearby intron sequences contain very high densities of sequences that enhance or reduce splicing and are recognised by a correspondingly large number of activator or repressor proteins. Without knowing the combinations, inter-dependence or dynamics of protein binding, we cannot understand the mechanisms by which the bound proteins affect events at the splice sites.

We will determine the patterns of protein binding and their subsequent mechanisms of action using our existing inter-disciplinary methodology and innovative approaches that we are uniquely suited to apply. We will use our single molecule (sm) microscopy methods (in routine use for a decade) to identify the heterogeneity, stoichiometry, co-occupancy, independence, permitted combinations and stability of complexes formed on SMN2 exon 7 and Bcl-X pre-mRNA in nuclear extracts (NE) when the exon/5' splice site is activated or repressed. The extent to which we can account for all proteins bound will be checked by a novel combination of sm fluorescence microscopy with interferometric scattering (iSCAMS) to measure the masses of the RNA complexes +/- 20 kDa. Stoichiometry and combination measurements may favour complex propagation or 3D-diffusion/RNA threading modes of action. The contribution of 3D interactions will be tested orthogonally by insertion of non-RNA linkers in splicing assays and novel bulky branches to block diffusion, measurement of RNA flexibility in NE by smFRET in vesicles, and proximity biotinylation in NE. The interactions of proteins (especially their flexible domains) with each other (by 3D or complex formation) and the RNA will be characterized by NMR in NE. Finally, real time observations of occupancy and changes in complex mass will be enabled by the development of new types of surface for sm work.

Planned Impact

1. Benefits to science in the UK.
UK frontier bioscience would be the major beneficiary of this research, for several reasons. (i) The transformative new understanding of one of the fundamental processes of life will have far-reaching implications for ways of thinking in gene expression and may even change the way in which these processes are represented in textbooks. (ii) The combination of new insights and the development of new methodology would provide a major boost to the reputation and morale of the UK RNA processing community. (iii) The application-driven developments in methodology will benefit innovatory science in a range of fields and improve the sustainability of the UK's position in the field. (iv) Academic and commercial scientists in this research area will benefit directly because we are keen to engage in collaborations using our methods and insights to develop a better understanding of the specific splicing switches that other researchers might be studying (relating, for example, to development or ageing).


2. Potential economic impact.
It is highly probable that there will be opportunities for health and UK bioscience industries. Splice site selection is fundamental to human health, and both ageing and many diseases (from neurodegeneration to cancer) are associated with, mediated by or caused by improper splice site selection. The first therapies based on modulating splicing are emerging, including most famously the first treatment for spinal muscular atrophy (SMA), but progress is limited by the lack of relevant knowledge about how to perturb the processes being modulated. This is illustrated well by the confusion as to the mechanisms of action of some of the small molecule therapies being developed for SMA. This means that effectively targeted or designed strategies are not generally an option. Understanding the molecular mechanisms of splicing will open up a new set of drug targets for rational development and lead to new classes of therapeutics. The life sciences sector has the largest involvement in R&D in the UK (>£4 billion) and employs >70,000, but there has been a decrease in large pharma employment recently and reduced investment in the most important area economically, small molecule drug discovery (while it has increased overseas; ABPI report, 2016; HMG Office for Life Sciences, 2016). Developing an understanding of the rules by which proteins modulate splicing will help in finding new targets for small molecules.

3. Benefits to science and the UK from training researchers with inter-disciplinary skills
This research will produce postdoctoral researchers trained in RNA biochemistry, chemical biology related to RNA, RNA and protein structural biology, single molecule methods in functional conditions and nanotechnology of surfaces, with knowledge of the broader contexts, new horizons and academic excellence. This is, of course, beneficial to their careers, but both fundamental science and the bioscience industries will benefit from young scientists who are used to thinking creatively and doing adventurous work and who can understand science outside their own original fields and see how inter-disciplinary collaborations enable it to be applied in new and exciting ways.

Publications

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Bunschoten RP (2024) Mechanistic Basis of the Cu(OAc)2 Catalyzed Azide-Ynamine (3 + 2) Cycloaddition Reaction. in Journal of the American Chemical Society

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Sperling J (2023) A cross-reactive plasmonic sensing array for drinking water assessment in Environmental Science: Nano

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Peschke F (2023) Glutathione Mediates Control of Dual Differential Bio-orthogonal Labelling of Biomolecules. in Angewandte Chemie (Weinheim an der Bergstrasse, Germany)

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Peschke F (2023) Glutathione Mediates Control of Dual Differential Bio-orthogonal Labelling of Biomolecules. in Angewandte Chemie (International ed. in English)

 
Title Raw figures from manuscript "Surface passivation with a perfluoroalkane brush improves the precision of single-molecule measurements" 
Description Raw data from mass photometer regarding the interaction of different biomolecules with new fluorous surfaces. The data showed that these surfaces almost completely avoid non-specific interactions and therefore could be ideal for single-molecule experiments. The file format is dictated by the supplier of Mass Photometer (Refeyn), and can be opened by Discover MP software. 
Type Of Art Film/Video/Animation 
Year Produced 2023 
URL https://leicester.figshare.com/articles/figure/Raw_figures_from_manuscript_Surface_passivation_with_...
 
Title Raw figures from manuscript "Surface passivation with a perfluoroalkane brush improves the precision of single-molecule measurements" 
Description Raw data from mass photometer regarding the interaction of different biomolecules with new fluorous surfaces. The data showed that these surfaces almost completely avoid non-specific interactions and therefore could be ideal for single-molecule experiments. The file format is dictated by the supplier of Mass Photometer (Refeyn), and can be opened by Discover MP software. 
Type Of Art Film/Video/Animation 
Year Produced 2023 
URL https://leicester.figshare.com/articles/figure/Raw_figures_from_manuscript_Surface_passivation_with_...
 
Description This award is still on-going. However, we have established that fluorous surfaces offer advantages for mass photometry but unfortunately are not suitable surfaces for single molecule microscopy because molecules diffuse rapidly in the surface - including molecules that were not designed to be tethered to the surface. This means that our SM microscopy of splicing reactions will have to be done on conventional surfaces, with all the attendant disadvantages. We have built a combined mass photometer/TIRF microscope and have shown that it can image single molecules in surfaces. The combination of two detection modes is clearly feasible, and will be the subject of an application for follow-on funding. The combination is the subject of a patent application. The detection of protein binidng to single molecules of RNA has revealed unexpected patterns of binding of two regulatory proteins, hnRNPG and Tra2beta, studies of which are ongoing, but it looks as though high levels of binding are cooperative and inhibit splicing. We have developed new software, FluoroTensor, for single molecule research. This incorporates two convolutional neural networks (a form of machine learning) and enables measurements of stoichiometry by step detection, step positioning, tracking and FRET analyses. This has been made freely available to the wider research community.
Exploitation Route FluoroTensor is going to be used widely in single molecule research, since it enables the most accurate step detection of any current methods, is comprehensive and there is an extensive user manual. The combination of mass photometry and TIRF microscopy is potentially powerful and we hope that follow-on funding will enable it to be developed commercially.
Sectors Digital/Communication/Information Technologies (including Software)

Manufacturing

including Industrial Biotechology

URL https://www.spliceselect.org
 
Title Development of new surfaces for use with iSCAMS (interference scattering mass spectrometry) 
Description The new surfaces permit many proteins or particles to dissociate instantly from the surface after initial contact. This will enable equilibrium measurements to be made with interacting macromolecules without the bias arising from preferential depletion of the larger molecules. In addition, measurements can be made with vesicles, which do not deposit and fuse with the surface. This will enable measurement of vesicles in a population. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact This method is an essential stepping stone for our later purposes. However, we intend to publish it soon and anticipate that it will be widely adopted by those engaged in single molecule research. 
 
Title Use of neural networks to detect single molecule bleaching steps of fluorescent proteins 
Description We have developed a new method for identifying bleaching steps with fluorescent proteins. Historically, this has been challenging because the fluorescence intensities vary widely from molecule to molecule when deposited on a surface, and our previous methods had replied on statistical analyses (Bayesian and Maximum Likelihood) that required checking by trained individuals. This new method will allow automatic assignments and the generation of additional data validating the assignments. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact This will dramatically improve the objectivity of our analyses and also increase the speed of our work very substantially. When published, the tools will be made freely available. 
 
Title Exon-independent recruitment of SRSF1 is mediated by U1 snRNP stem-loop 3 
Description Collection of single molecule imaging data supporting the publication with this title in EMBO Journal in 2022. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Data produced findings described in the EMBO J. paper. 
URL https://doi.org/10.25392/leicester.data.c.5521944.v1
 
Title A microscopy system 
Description An interferometric scattering microscopy system for inspecting a sample comprising a fluorescently labelled component, the system comprising: a sample holder mount configured to mount a waveguide sample holder comprising: a first surface for positioning the sample; and a second surface opposite the first surface; a coupling arrangement configured to couple a fluorescence excitation light field to the waveguide sample holder to guide the fluorescence excitation light field along the waveguide sample holder by total internal reflection from the first surface and the second surface; an interferometric scattering microscope arranged to: illuminate the sample with a source light field; and detect interference between: a reference light field comprising a reflection of the source light field; and a scattered light field comprising scatter of the source light field from the sample; and a fluorescence microscope arranged to collect a fluorescence emission light field emitted from the fluorescently labelled component in response to absorption of the fluorescence excitation light field. 
IP Reference 2307933.8 
Protection Patent / Patent application
Year Protection Granted
Licensed No
Impact Patent applied for; licensing will be associated with our application for follow-on funding
 
Title FluorTensor 
Description The identification of photobleaching steps in single molecule fluorescence imaging is a well-established procedure for analysing the stoichiometries of molecular complexes. Nonetheless, the method is challenging with protein fluorophores because of the high levels of noise, rapid bleaching and highly variable signal intensities, all of which complicate methods based on statistical analyses of intensities to identify bleaching steps. It has recently been shown that deep learning by convolutional neural networks can yield an accurate analysis with a relatively short computational time. We describe here an improved use of such an approach that detects bleaching events even in the first time point of observation, and we have included this within an integrated software package incorporating fluorescence spot detection, colocalisation, tracking, FRET and photobleaching step analyses of single molecules or complexes. This package, known as FluoroTensor, is written in Python with a self- explanatory user interface. 
Type Of Technology Software 
Year Produced 2024 
Open Source License? Yes  
Impact Considerable improvement in accuracy and ease of measuring stoichiometries in single molecule experiments using photobleaching; should be widely used. 
URL https://www.spliceselect.org/wp-content/uploads/2024/02/FluoroTensor-v6.6.8r-Full-User-Guide.pdf
 
Description Invited seminar in Technical University, Munich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Visit to lab of Prof. Sattler to present recent research on splicing. About 15 members of his and related groups attended. Considerable interest and lively discussions.
Year(s) Of Engagement Activity 2022
 
Description Poster at SpliceCon 2021 meeting (RNA Society Steenbock Symposium) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presented by postdoc SG.
Year(s) Of Engagement Activity 2021
URL https://www.rnasociety.org/splicecon-2021--a-steenbock-symposium
 
Description Presentation at BBSRC workshop for sLoLa grant-holders 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Description of the sLoLa and its achievements thus far.
Year(s) Of Engagement Activity 2022
 
Description Presentation to London RNA Club 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact Seminar to London RNA Club
Year(s) Of Engagement Activity 2022
 
Description Public lecture - broadcasted and published online 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This public lecture was hosted by the University of Leicester as part of its Centenary Year celebrations. Invitations were sent to alumni and supporters of the University, current undergraduate and postgraduate students and research staff, along with academic colleagues from other institutions.
Year(s) Of Engagement Activity 2021
URL https://www.youtube.com/watch?v=wja7RvSfikE
 
Description Selected talk at SpliceCon virtual meeting (RNA Society Steenbock Symposium) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk to international splicing meeting selected from abstracts
Year(s) Of Engagement Activity 2021
URL https://www.rnasociety.org/splicecon-2021--a-steenbock-symposium
 
Description Talk at UK RNA Splicing Workshop, January 2021 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk at meeting by myself including new data about the interaction of SRSF1 and U1 snRNP, major determinants of splice site selection. This raised the national profile of our work, even though it has been grievously hindered by Covid-19.
Year(s) Of Engagement Activity 2021
 
Description Talk by Dr Bueno Alejo at 28th IUPAC international symposium in photochemistry 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation of work on fluorous surfaces at meeting sparked interest in further developments and use of our new method
Year(s) Of Engagement Activity 2022
 
Description Talk by Dr Bueno Alejo at mass photometry user meeting, Oxford 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Meeting regarding uses of mass photometry
Year(s) Of Engagement Activity 2022
 
Description Talk by Dr Hesna Kara at annual NMR meeting in Parpan, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk about new methods using fluorinated RNA at annual meeting.: 2'-19F labelling of ribose in RNAs, a tool to analyse RNA/protein interactions by NMR
Year(s) Of Engagement Activity 2023
 
Description Talk by Dr Santana Vega to SPIE Photonics West conference, San Francisco 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Title: A new platform for single molecule measurements using the fluorous effect.

Awarded the prize for the best talk by a young investigator
https://www.picoquant.com/scientific/young-investigator-award#:~:text=Young%20scientists%20are%20encouraged%20to,cash%20award%20worth%20750%20USD.&text=Adam%20Bowman%2C%20Stanford%20University%20(USA,of%20single%20molecules%20and%20neurons%22.
Invited to talk in September at 28th International Workshop on "Single Molecule Spectroscopy and Super-resolution Microscopy"
Year(s) Of Engagement Activity 2022
URL https://spie.org/photonics-west/presentation/A-new-platform-for-single-molecule-measurements-using-t...
 
Description Talk by Max Wills at UK RNA Splicing meeting, Rydal, Jan. 2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Max Wills is a PhD student engaged in sLoLa work and funded by the university as part of its support for the sLoLa. He described his use of convolutional neural networks for the analysis of single molecule data, which created much interest.
Year(s) Of Engagement Activity 2023
 
Description Talk by Prof. Burley at Sheffield 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited talk at Sheffield University
Year(s) Of Engagement Activity 2022
 
Description Talk by Prof. Burley at University of Wollongong 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited talk to university department
Year(s) Of Engagement Activity 2022