Post transcriptional regulatory networks

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

Like all cells in the body, brain cells must rely on the same set of genes - about 25,000 - as the blueprint for the unique assortment of proteins they need to carry out their function. One way cells achieve this diversity is via a process called RNA splicing. This process allows the initial RNA copy of the gene to be cut into pieces, some of which are used to produce a mature mRNA that codes for cellular proteins. In alternative splicing, pieces of the pre-mRNA are stitched together in different ways to produce different mRNAs. By regulating alternative splicing, cells can produce a wide variety of proteins from a finite number of genes. This capacity is believed to be critical for the complex workings of human cells, especially neurons in the brain.
The job of regulating splicing is done by proteins that bind the RNA. Defects in such proteins underlie several human brain disorders, but the RNA targets of these proteins are mostly unknown. We use a new type of a microarray that was designed to detect changes in alternative splicing. By comparing the RNA molecules found in normal tissue with those of diseased tissue, or tissue lacking a specific RNA-binding protein, we can identify mRNAs that are misregulated when a normal regulatory mechanism is disrupted in the brain.
We have also developed a method, termed iCLIP, which allows us to catalogue complete lists of RNAs that are regulated by specific RNA binding proteins. iCLIP uses a unique combination of biochemical techniques, including photo cross-linking, immunoprecipitation, protein electrophoresis and ultra-high throughput sequencing. We use iCLIP to understand how mutations disrupt the regulatory networks, and how the resulting aberrant splicing of RNAs leads to neurologic disorders.

Technical Summary

Before being translated into a protein, an mRNA traverses different cellular compartments and undergoes an array of processing and regulatory steps. These steps are regulated mainly by RNA-binding proteins, which interact with the cis-acting elements in pre-mRNAs and mRNAs. Most of the cis-acting elements reside in non-coding RNA regions; whereas less than 1% of human genome corresponds to the protein-coding sequence, over 25% corresponds to introns and untranslated mRNA regions. We aim to elucidate the functions of these non-coding RNA regions by studying their interactions with RNA-binding proteins.
To characterize the functions of cis-acting RNA elements, it is crucial to examine the regulatory protein-RNA interactions in the intact cells and tissues. For this purpose, we developed individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP), which exploits high-throughput sequencing to identify the in vivo protein-RNA crosslink sites. The high resolution and quantitative nature of iCLIP allows us to monitor the dynamic composition of ribonucleoprotein complexes. This enables us to investigate the regulatory mechanisms that involve competitive or cooperative interactions between RNA and multiple proteins. We examine the role of RNA-binding proteins in regulating alternative splicing, 3' end processing or mRNA localization. We integrate these functional studies with the position of protein-RNA interactions determined by iCLIP, which often reveals general positional principles underlining post-transcriptional regulation.
We are particularly interested in the importance of post-transcriptional regulation for neuronal development. The extreme cell polarity poses specific problems for neurons, which are often solved with the use of a complex and coordinated RNA regulation. We study how the regulation of pre-mRNA processing is coordinated with neuronal mRNA localisation and translation. In addition, we inquire how genomic variation within the non-coding RNA elements affects post-transcriptional regulation in the induced pluripotent stem (iPS) cells from different individuals, and how defective post-transcriptional regulation can lead to neurologic disorders.
 
Description EC ITN Grant
Amount € 261,201 (EUR)
Funding ID FP7-people-2011-ITN-289007 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 01/2011 
End 10/2015
 
Description ERC Grant
Amount £819,224 (GBP)
Funding ID ERC-2007-StG-206726 - CLIP 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2008 
End 08/2013
 
Description HFSP Grant
Amount £137,969 (GBP)
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 10/2008 
End 09/2011
 
Description HFSP Long-term Fellowship
Amount £86,830 (GBP)
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 10/2009 
End 09/2011
 
Description MRC Fees Only Studentship
Amount £58,500 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description NIH Grant
Amount £166,346 (GBP)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 10/2008 
End 09/2011
 
Description Nakajima Foundation Scholarship
Amount £45,000 (GBP)
Organisation Japan Student Services Organization (JASSO) 
Department Heiwa Nakajima Foundation Scholarships
Sector Charity/Non Profit
Country Japan
Start 10/2010 
End 09/2013
 
Description Synergia grant
Amount £200,000 (GBP)
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 12/2010 
End 12/2013
 
Description Wellcome Trust/MRC Neurodegenerative Diseases Initiative
Amount £123,586 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2010 
End 03/2015
 
Title ASPIRE software 
Description ASPIRE software for analysis of splice-junction microarrays from Affymetrix. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact The software is used to analyse data of several research groups 
 
Title iCLIP method 
Description new methods for identifying protein-RNA in live cells and tissues, which involve UV-crosslinking and immunoprecipitation method (CLIP) followed by multi-step preparation of cDNA libraries for high-throughput sequencing 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact The method has been adopted by over 100 labs, cited ~800 times so far, and 18 further variant methods have been published by multiple labs. We have continually been improving it and writing methods and review articles to keep the field well informed and integrated. 
URL https://www.sciencedirect.com/science/article/pii/S1097276518300054
 
Title iCount 
Description A databaset and online server for analysis of iCLIP data 
Type Of Material Improvements to research infrastructure 
Year Produced 2010 
Provided To Others? Yes  
Impact Analysis of data that contributed to several publications. 
URL https://github.com/tomazc/iCount
 
Description A systematic and functional analysis of HIF-dependent splice regulator expression and pre-mRNA alternative splicing in pathologic stress-induced cardiac hypertrophy. 
Organisation ETH Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution iCLIP and microarray analyses.
Collaborator Contribution Provided samples and data.
Impact This resulted in a joint Nature paper.
Start Year 2010
 
Description Analysis of iCLIP data using iCount server. 
Organisation University of Ljubljana
Department Faculty of Computer and Information Science
Country Slovenia 
Sector Academic/University 
PI Contribution We produce the iCLIP data and discuss the software.
Collaborator Contribution iCount server is required for analysis of iCLIP data.
Impact Two joint publications published, several more in review or in preparation.
Start Year 2008
 
Description RNA Processing proteins and neurodegeneration: exploring mechanisms and modelling disease 
Organisation King's College London
Department MRC Centre for Neurodegeneration Research
Country United Kingdom 
Sector Public 
PI Contribution iCLIP with TDP-43, analyses of splicing in human brain.
Collaborator Contribution Provided samples and shared unpublished data.
Impact Two joint publications resulted from this, the primary one in Nature neuroscience.
Start Year 2009
 
Description Structure and dynamics of neuronal granules that regulate RNA localization. 
Organisation Medical University of Vienna
Department Center for Brain Research
Country Austria 
Sector Academic/University 
PI Contribution Study of neuronal RNA-binding proteins.
Collaborator Contribution Contributed to joint publications, provided reagents and shared data.
Impact Several manuscripts resulted from this work, including Sugimoto et al, Nature, 2015.
Start Year 2008
 
Description splice-junction microarray data analysis 
Organisation Affymetrix Inc.
Country United States 
Sector Private 
PI Contribution We wrote software for analysis of new Affymetrix splice-junction microarrays.
Collaborator Contribution 50 free microarrays.
Impact 50 free microarrays were used for the first 5 manuscripts published by my group.
Start Year 2006
 
Title An improved method to detect protein-RNA interactions 
Description The patent describes the individual-nucleotide resolution CLIP (iCLIP) 
IP Reference Insufficient Information 
Protection Patent application published
Year Protection Granted
Licensed No
Impact Two publications so far.
 
Description Genome-wide mapping of protein-RNA interactions 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Pre-mRNA processing and disease, EMBO workshop, Cortina d'Ampezzo, Italy
Swiss RNA workshop, Bern, Switzerland
EMBO European Life Sciences meeting, Dresden, Germany


made new contacts
Year(s) Of Engagement Activity 2007
 
Description Invited presentations at multiple conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Therapeutic Strategies Targeting The Spiceosome, SMSdrug meeting, London, UK
The Complex life of mRNA, EMBO Symposium, Heidelberg, Germany
Interactions of signaling pathways and non-coding RNA, EMBO workshop, Switzerland
Neurons Under Stress, CSND meeting, Dublin, Ireland
Neurotrophic factors and Plasticity of the Nervous system, Wurzburg, Switzerland
Cell Dynamics and Disease, CEDAD Graduate School Meeting, Muenster, Germany
Visualizing Biological Data, EMBO Conference Series, EMBL, Heidelberg, Germany
SA Society of Biochemistry and Molecular Biology meeting, Drakensberg, South Africa
Year(s) Of Engagement Activity 2012
 
Description Invited presentations at multiple conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact New methods in neuroscience, SfN meeting, Washington, USA
RNA-binding proteins in neurodegeneration, Brain Research conference, Washington, USA
Unanswered questions in transcription, CRI International Symposium, Cambridge, UK
Czech RNA Society meeting, Ceské Budejovice, Czech Republic
International Workshop on Genomics of Health and Diseases, Natal, Brazil
Year(s) Of Engagement Activity 2011
 
Description Invited presentations at multiple conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Translational Control, EMBO Symposium, Heidelberg, Germany
Biomolecular Structure and Mechanism, ETH student Symposium, Zurich, Switzerland
RNA society meeting 2013, Davos, Switzerland
UK RNA splicing workshop", Lake District, UK
Advances in molecular mechanisms underlying neurological disorders, Neurochemistry conference, Bath, UK
RNA Transport, 107th International Titisee Conference, Titisee, Black Forest, Germany
Year(s) Of Engagement Activity 2013
 
Description Invited presentations at multiple conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact RNA Mis-Splicing: Disease and Therapeutics, Eurasnet meeting, Cambridge UK
Year(s) Of Engagement Activity 2010
 
Description Organising roles in scientific meetings 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organiser of the "RNA dynamics in development and disease" EMBO workshop, Ljubljana, Slovenia, 2019
Session chair of "New Research Trends and Technologies", RNA society meeting, Cracow, Poland, 2019
Session chair of "Transcriptomics and Bioinformatics of Splicing", Riboclub, Cheribourg, Canada, 2018
Session chair of "Transcription and Translation", Wellcome Trust meeting, London, UK, 2018
Co-organiser of "Protein-RNA Interactions", Keystone symposium, Banff, Canada, 2017
Co-organiser of "iCLIP: Genomic views of protein-RNA interactions", EMBO, Germany, 2015 & 2017
Session chair of "Alternative splicing", RNA society meeting, Madison, USA, 2015
Session chair of "Protein-RNA interactions", Eukaryotic mRNA Processing, CSH meeting, CSHL, USA, 2015
Chair of "RNA bioinformatics" session at the "RNA society meeting", Davos, Switzerland, 2013
Chair of "Protein-RNA interactions" at the "Translational control" EMBO meeting, Heidelberg, 2013
Chair of "Transcriptome level analysis of splicing" session at the "UK RNA splicing workshop", UK, 2013
Co-organiser of "RNA-binding proteins in development and cancer", Baeza, Spain, 2009
Year(s) Of Engagement Activity 2009,2013,2015,2017,2018
 
Description RNA-protein interactions in neurodegeneration 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The talk was at MRC Centre for Neurodegeneration Research, London, and initiated a fruitful collaboration

publication in preparation
Year(s) Of Engagement Activity 2008
 
Description invited presentations at multiple conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact New methods in neuroscience, SfN meeting, Washington, USA
RNA-binding proteins in neurodegeneration, Brain Research conference, Washington, USA
Unanswered questions in transcription, CRI International Symposium, Cambridge, UK
Czech RNA Society meeting, Ceské Budejovice, Czech Republic
International Workshop on Genomics of Health and Diseases, Natal, Brazil
Year(s) Of Engagement Activity 2011
 
Description invited talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Slovenian Biochemical Society meeting, Novo Mesto, Slovenia
Gene expression in neuronal disease, UK Biochemical Society meeting, Bristol, UK
Alternative Splicing - Special Interest Group Meeting, Stockholm, Sweden
Aberrant Splicing in Neuromuscular and Neurodegenerative Processes, Eurasnet meeting, Trieste, Italy

initiated new collaborations
Year(s) Of Engagement Activity 2009