Novel functions of alternative pre-mRNA splicing coupled with nonsense-mediated decay

Lead Research Organisation: King's College London
Department Name: Developmental Neurobiology

Abstract

Precise regulation of cell- and tissue-specific genes is indispensable for proper development and function of our organism. Importantly, abnormal gene expression has been linked to devastating neurological and neuropsychiatric diseases such as myotonic dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, Alzheimer's disease and dementias. Yet another example of potentially dire consequences caused by defective genes is provided by a range of hereditary disorders affecting multiple tissues and organs. Many of these conditions are associated with considerable patient suffering and mortality and constitute a serious burden for the national health system.

Expression of our genes proceeds as a succession of distinct molecular steps that include transcription, processing of primary transcripts into messengers, and ultimately translation of the messengers into proteins. Several control mechanisms operating at each of these steps ensure that protein products of our genes appear in the right place and at the right time. One such control called nonsense-mediated decay, or NMD, eliminates messengers with defective protein-coding capacity thus allowing the cell to cope with deleterious mutations and occasional errors in primary transcript processing. Surprisingly, recent studies suggest that this mechanism is also used to regulate gene expression under normal circumstances. In this case, corresponding primary transcripts can be processed to generate two or more alternative messenger variants, with some variants encoding proteins and the others degraded by NMD. Several genes indispensible for our nerve cells appear to be regulated in this manner and additional research in this field promises further exciting discoveries.

Here we propose to uncover novel genes that are controlled by alternative processing/NMD during brain development and to begin understanding their functional significance. We will advance our research by combining molecular, cellular and developmental biology techniques with advanced bioinformatics approaches. Our work will have important biomedical implications. Indeed, at least one novel gene, Hps1, identified in our preliminary experiments has been directly linked to a hereditary disease called Hermansky-Pudlak Syndrome and understanding regulation of this gene will likely provide important insights into this and a large group of other metabolic disorders associated with similar molecular defects. Wider medical relevance of our study will be guaranteed by its general relevance to neurological and neuropsychiatric diseases associated with aberrant regulation of genes.

Technical Summary

An important transcriptome-wide event in differentiating neurons involves coordinated down-regulation of multiple genes expressed at high levels in neural precursors (NPs). Here we will investigate a novel mechanism mediating this transition by switching alternative splicing (AS) patterns of NP-specific transcripts and triggering their nonsense-mediated decay (NMD). We hypothesise that RNA-binding protein Ptbp1 that is expressed at high levels in NPs but down-regulated in neurons, may coordinate a large part of this AS-NMD program. Indeed, our preliminary studies have begun uncovering genes expressed at optimal level in the presence of Ptbp1 but undergoing NMD in its absence.

To further test our hypothesis, we will first identify corresponding target genes in mouse neuroblastoma cells by transcriptome-wide RNA sequencing followed by advanced bioinformatics interpretation of condition-dependent changes in the numbers of exonic, intronic and splice junction RNA-seq reads. We will validate neuroblastoma data and refine our understanding of the Ptbp1/AS-NMD network using an in vitro neurogenesis model, primary cultures of neural stem cells and neurons and ex vivo neural tube explants. Finally, we will elucidate biological significance of the most promising Ptbp1/AS-NMD targets in cell lines and primary cultures through a combination of molecular, cellular and developmental biology approaches.

By focusing on a novel class of Ptbp1/AS-NMD targets and performing differentiation stage-resolved analyses the proposed program will be poised to generate valuable insights into fundamental mechanisms underlying brain development and function. Importantly, at least one Ptbp1-activated AS-NMD target, Hps1, has been linked to a hereditary condition called Hermansky-Pudlak Syndrome. Wider medical implications of our study will be guaranteed by its general relevance to neurological and neuropsychiatric diseases associated with aberrant regulation of genes.

Planned Impact

We anticipate that the proposed programme will generate considerable economic and societal impacts by contributing to medicine, education and staff training.

Medicine
Underscoring medical significance of our work, many human diseases are associated with defects in posttranscriptional control of gene expression. Importantly, RNA-based processes are deregulated in several devastating neurological and neuropsychiatric disorders including myotonic dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, frontotemporal dementia, and possibly Alzheimer's and Huntington's diseases. These conditions accompanied by considerable patient morbidity and mortality also account for an extremely large fraction of the annual £112 billion cost of brain disorders in the UK (J. Psychopharmacol. 2013, 27:761-770). By shedding light on important aspects of RNA metabolism in the nervous system context the proposed programme should improve our understanding of molecular aetiology of these and other diseases and ultimately lead to developing advanced therapies and diagnostic tools. Our work will also have another important biomedical dimension. At least one gene identified in our preliminary experiments, Hps1, has been directly linked to Hermansky-Pudlak Syndrome, a hereditary disease related to a larger cohort of lysosomal storage diseases occurring with collective incidence of ~1 in 7000-8000 live births (Biochem. Soc Trans. 2000, 28:150-154). New insights into Hps1 regulation generated by our program may eventually improve the way these diseases are detected and treated in clinical settings. Although materialisation of these important benefits may require years and possibly decades, we are convinced that contribution of our work to this process will be substantial.

Education
Our work will rely on a multidisciplinary strategy combining bioinformatics and systems biology with more traditional approaches. This trend is becoming increasingly prevalent in life sciences thus necesitating corresponding updates to secondary and tertiary science education. By reaching out to school and undergraduate students we hope to generate valuable experience that may impact future long-term changes in the education sector. Of note, a number of undergraduate students successfully completed short, typically 2-12 month research projects in PI's lab in the past. In 2015-2017, the lab is expected to host students from the Judd School (Tonbridge, Kent) and KCL undergraduates. We plan to engage these students in experimental and computational biology projects outlined in this proposal, which will provide them with first-hand experience in multidisciplinary research. We will additionally use our methodology and data as a teaching device in lectures and tutorials for KCL undergraduate and graduate students.

Staff training
The proposed project will provide a natural framework for training of a postdoctoral fellow who will master a wide range of molecular biology, cell engineering, developmental neurobiology and bioinformatics techniques and will additionally acquire advanced communication and managerial skills. This comprehensive training will maximise his or her value as a skilled employee capable of making important contributions to the UK academia and industry within 3-6 years since the start of the programme. Moreover, by interacting with other lab members the postdoctoral fellow will undoubtedly facilitate their professional growth.

Publications

10 25 50
 
Description As a part of our research program we systematically analyzed splice isoforms expressed at an optimal level only in the presence of an RNA-binding protein called PTBP1. We carried out extensive molecular characterization of several targets of the alternative splicing - nonsense mediated decay (AS-NMD) pathway and are currently preparing a manuscript describing these analyses. Briefly, our work suggests that regulation of gene expression by PTBP1/AS-NMD in developing brain is substantially more widespread than currently thought. We also identified an interesting category of PTBP1 targets that show a tendency for increased co-expression of their alternatively spliced variants during neuronal development. We showed that this results in stable co-expression of two distinct splice isoforms of the cell polarity factor Cdc42 and that both of these isoforms are essential for correct neuronal development. Our work describing this finding was published in Cell Reports (2016) and highlighted in our review in Biochemical Society Transactions (2016) as well as several science news websites.

We also identified a new repeat-containing long-noncoding RNA, PNCTR, that controls PTBP1 activity in cancer cells. Importantly, we showed that this RNA promotes cancer cell survival at least in part by regulating expression levels of a specific AS-NMD target of PTBP1 encoding a checkpoint kinase, CHEK2. Results of this study have been published in a reputable scientific journal (Molecular Cell 2018) and highlighted in a press-release article published in the same issue. Relevant to our RNA biology interests in general and this project in particular, we additionally participated in collaborative work aiming to delineate mechanisms underlying evolution of new properties in protein families involved in eukaryotic RNA metabolism. One important conclusion emerging from this study so far is that functional innovation in evolutionarily related RNA metabolic enzymes may occur through relatively minor alterations in their protein folds and quaternary structures (JBC 2016). This grant also helped us make important contributions to understanding regulation of RNA splicing in human and mouse cells (two publications in Nucleic Acids Research and one in PLoS Genetics in 2017). Finally, we used bioinformatics tools developed as a part of our BBSRC-supported work to help our colleagues to illuminate molecular mechanisms contributing to a heart disorder called hypertrophic cardiomyopathy (collaborative paper in Journal of Molecular and Cellular Cardiology 2018).
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Exploitation Route Our findings so far illuminate important aspects of post-transcriptional gene regulation in mammalian nervous system and will be useful to other scientists who study related problems in other biological systems. This work may also be used as teaching material for undergraduate and PhD students attending lectures, tutorials and seminars on gene regulation mechanisms and neurobiology.
Sectors Education,Healthcare

URL https://www.sciencedaily.com/releases/2016/04/160428131939.htm
 
Description We used research program supported by this grant as a framework for training of four KCL postgraduate students rotating in our lab for 3 months each (part of the KCL curriculum) and two summer undergraduate students (outside of the KCL curriculum). One of these summer projects was supported by a prestigious Wellcome Trust Biomedical Vacation Scholarship. The grant also allowed us to host a 2-month summer project for a student from the Judd School, Tonbridge, Kent, a 2.5-month project of a summer student from University California Irvine visiting KCL as a part of the Minority Science Program, as well as bioinformatics training for four postgraduate students from Estonia who visited us in November 2018-March 2019. To maximize societal impact of our work, we contributed to press-releases explaining significance of our findings to a wide audience on various websites. The impact of our work has been further widened by a preview article in Molecular Cell (https://www.ncbi.nlm.nih.gov/pubmed/30388407) showcasing our paper on repeat-containing noncoding RNAs and one of their AS-NMD targets (https://www.ncbi.nlm.nih.gov/pubmed/30318443). We also participated in organization of an international symposium on gene expression in health and disease, which was held in Tallinn, Estonia, March 16, 2018. This event provided an excellent opportunity for UK-based participants as well as international attendees to enhance their presentation and networking skills. We also presented our study on repeat-containing noncoding RNAs and AS-NMD in this meeting.
First Year Of Impact 2016
Sector Education
Impact Types Cultural,Societal

 
Description Marie Sklodowska-Curie Postdoctoral Fellowship
Amount £128,418 (GBP)
Funding ID 751739 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 05/2017 
End 10/2019
 
Description Research and Innovation Staff Exchange (RISE)
Amount £1,117,464 (GBP)
Funding ID 734791 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2017 
End 12/2020
 
Title New method to quantify co-expression of alternatively processed messenger RNA isoforms 
Description As a part of our project, we introduced a novel statistic called isoform co-expression index. It allows one to quantify co-expression of alternatively processed messenger RNA isoforms in any biological sample, from an individual cell to entire organism. We published this new approach in Cell Reports in 2016. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2016 
Provided To Others? Yes  
Impact The expected beneficiaries of this innovation will be researchers investigating alternative splicing in different biological contexts. 
URL http://www.cell.com/cell-reports/comments/S2211-1247(16)30411-9
 
Description Developing an AS-NMD analysis pipeline 
Organisation University of Tartu
Country Estonia 
Sector Academic/University 
PI Contribution Participated in development of an R/BioConductor package for discovery of new genes regulated by alternative splicing coupled with nonsense-mediated decay (AS-NMD)
Collaborator Contribution Participated in development of an R/BioConductor package for discovery of new genes regulated by alternative splicing coupled with nonsense-mediated decay (AS-NMD)
Impact A prototype package developed. This multi-disciplinary activity combines our expertise in molecular biology with extensive bioinformatics expertise of the BIIT group, Tartu University (https://biit.cs.ut.ee/).
Start Year 2018
 
Description Evolution of eukaryotic RNA-interacting proteins 
Organisation Nanyang Technological University
Country Singapore 
Sector Academic/University 
PI Contribution This collaboration aims to understand mechanisms underlying evolution of new properties in eukaryotic enzyme families involved in RNA metabolism. My lab has contributed to this effort by analyzing phylogeny and biochemical properties of a representative group of these proteins.
Collaborator Contribution Our collaborators determined crystal and electron microscopy structures of orthologously related RNA metabolic enzymes and predicted how structural differences may lead to functional innovation. They are currently working on structural characterization of complexes between these proteins and their RNA ligands.
Impact Results of this multidisciplinary collaboration were published in the following article: Qian X, Hamid FM, El Sahili A, Darwis DA, Wong YH, Bhushan S, Makeyev EV*, Lescar J* .J Biol Chem. 2016 Apr 22;291(17):9295-309. doi: 10.1074/jbc.M115.685933. Epub 2016 Feb 23. (*= co-corresponding authors). Relevant research disciplines include biochemistry, molecular biology, structural biology and evolutionary biology.
Start Year 2015
 
Description Regulation of pre-mRNA splicing by RNA-binding protein RBM10 
Organisation University College London
Department Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in bioinformatics and minigene splicing assays and general knowledge of splicing regulation pathways.
Collaborator Contribution Expertise in structural biology and knowledge of RNA-binding protein activities.
Impact Published the following paper: Collins KM, Kainov YA, Christodolou E, Ray D, Morris Q, Hughes T, Taylor IA, Makeyev EV*, Ramos A*. (2017) An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion. Nucleic Acids Res. 2017 Apr 4. doi: 10.1093/nar/gkx225. (*= Co-corresponding authors.)
Start Year 2016
 
Description Role of alternative splicing and quality control mechanisms in regulation of minor spliceosome activity 
Organisation University of Helsinki
Department Institute of Biotechnology
Country Finland 
Sector Academic/University 
PI Contribution Expertise in alternative splicing, RNA quality control pathways and culturing primary neural cells.
Collaborator Contribution Expertise in biology of the minor spliceosome.
Impact Published the following paper: Verbeeren J, Verma B, Niemelä EH, Yap K, Makeyev EV, Frilander MJ.(2017) Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.PLoS Genet. 2017 May 26;13(5):e1006824. doi: 10.1371/journal.pgen.1006824.
Start Year 2016
 
Description Role of mutant CSRP3 protein in cardiomyopathy 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We used bioinformatics pipelines developed as a part of our BBSRC-supported research to understand molecular mechanisms linking mutations in the cysteine and glycine rich protein 3 (CSRP3) with hypertrophic cardiomyopathy.
Collaborator Contribution Our collaborators developed a mouse model for hypertrophic cardiomyopathy (HCM) and carried out its extensive phenotypic characterization at the Division of Cardiovascular Medicine, Radcliffe Department of Medicine, Oxford University. Data obtained as a result of these efforts suggest that reduced levels of functional CSPR3 may be a common mechanism underlying HCM.
Impact Results of this collaboration have been published in the following open-access paper: J Mol Cell Cardiol. 2018 Aug;121:287-296. doi: 10.1016/j.yjmcc.2018.07.248. Epub 2018 Jul 23. (https://www.jmmc-online.com/article/S0022-2828(18)30692-8/fulltext)
Start Year 2018
 
Description Bioinformatics training for international students 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We provided training in bioinformatics to 4 post-graduate students from Estonia who visited us for 1-2 month each as a part of SZ-TEST exchange program. Although the actual visits were funded by another grant (Marie Sklodowska-Curie Actions Research and Innovation Staff Exchange), we used bioinformatics tools developed as a part of our BBSRC-supported projects for training purposes.
Year(s) Of Engagement Activity 2018,2019
URL https://sztest.eu/
 
Description Hosting a Wellcome Trust-supported undergraduate project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact We hosted a 2-month research project for an undergraduate student from King's College London supported by a Wellcome Trust Biomedical Vacation Scholarship. The activity provided the student with an opportunity to apply for research funding and acquire a set of practical skills in molecular and cellular biology. We are convinced that this experience promoted student's interest in science and expanded his career possibilities in this field.
Year(s) Of Engagement Activity 2018
 
Description Hosting a summer research project for a student from University California Irvine 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact We hosted a summer research project for an undergraduate student from University California Irvine participating in a Minority Science Program. The purpose of this activity was to allow the student to participate in biomedical research and learn new molecular and cellular biology techniques. The student enjoyed his experience and subsequently presented the results obtained in our lab in several meetings including an AAAS Conference in 2017 (https://aaas.confex.com/aaas/2017/webprogram/Paper20152.html) and Minority Science Program meeting in 2016 (https://www.bio.uci.edu/events/2016-minority-science-programs-msp-research-symposium-day-1/). This experience encouraged the student to continue his scientific education at the postgraduate level.
Year(s) Of Engagement Activity 2016
URL https://www.bio.uci.edu/events/2016-minority-science-programs-msp-research-symposium-day-1/
 
Description Hosting an undergraduate summer research project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact We hosted a 2-month research project for an undergraduate student from King's College London. This activity provided the student with an opportunity to participate in biomedical research and also allowed him to acquire a set of practical skills in molecular and cellular biology. We are convinced that this experience promoted student's interest in science and expanded his career possibilities in this field.
Year(s) Of Engagement Activity 2017
 
Description Hosting research projects for students from the Judd School, Kent 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Our lab hosted 2-month research projects for two Judd pupils. This expanded pupils' knowledge in molecular and cellular biology and provided them with a unique opportunity to participate in biomedical research. We are convinced that this experience promoted pupils' interest in science and technology and expanded their possibilities for further education in this area.
Year(s) Of Engagement Activity 2015,2016
 
Description Organization of symposium on gene expression in health and disease 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We co-organized a one-day symposium on "Gene expression in health and disease", an event designed to allow mainly students and postdocs to present their research data to an international audience from Estonia Finland and the UK. Three postdoctoral fellows involved in our BBSRC-supported research have been selected to give 15-min presentations. This is an excellent framework for sharing scientific knowledge, fostering future collaborations and improving presentation skills of young scientists.
Year(s) Of Engagement Activity 2018
 
Description Participation in "Glow in the dark science" public outreach program in London primary schools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This project is a part of the British Science Week event (https://www.britishscienceweek.org/). It introduces primary school children to science in a playful manner. The project is based on activity stations where the pupils learn the basis of how fluorescence works and observe fluorescently labelled fish and fruit flies, among others. Most pupils attending this event clearly enjoyed the program and asked relevant questions. We believe this project should helped the pupils develop interest in life sciences and biomedical medical research.
Year(s) Of Engagement Activity 2016,2017,2018
URL https://devneuro.org/cdn/news-detail.php?NewsID=210&type=91
 
Description Participation in DevNeuro Academy 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Members of the Makeyev lab participated in the DevNeuro Academy project consisting of a regular program of activities designed to improve the progression and success of school students currently under-represented at our university and other institutes of higher education. The project combines a series of four in-school interactive 'Discovery workshops' with a two-week laboratory summer research work experience at the Centre for Developmental Neurobiology, KCL.
Year(s) Of Engagement Activity 2018
URL https://devneuro.org/cdn/public-engagement-dna.php
 
Description Press release article by Molecular Cell highlighting our publication 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact A highlight article showcasing our publication in the same issue of Molecular Cell
Year(s) Of Engagement Activity 2018
URL https://www.ncbi.nlm.nih.gov/pubmed/30388407
 
Description Press release for the Cell Rep. 2016 publication 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact We participated in a press release for our Cell Reports paper explaining significance of this work to the general public. The press release was published on the following science news websites:
https://www.sciencedaily.com/releases/2016/04/160428131939.htm
https://www.eurekalert.org/pub_releases/2016-04/kcl-coa042016.php
https://medicalxpress.com/news/2016-04-co-expression-alternative-gene-products-neurons.html
https://www.follownews.com/coexpression-of-alternative-gene-products-helps-neurons-take-shape-16knr
http://braintoolsnews.com/wp/feed-items/co-expression-of-alternative-gene-products-helps-neurons-take-shape-2/
http://www.hitechdays.com/news/8570/co-expression-of-alternative-gene-products-helps-neurons-take-shape/
https://scifeeds.com/news/co-expression-of-alternative-gene-products-helps-neurons-take-shape/
Year(s) Of Engagement Activity 2016
 
Description Press release on a KCL website 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We participated in a drafting a press release highlighting significance of our work. The press release was published on the Centre for Developmental Neurobiology website:
https://devneuro.org/cdn/news-detail.php?NewsID=298&type=93
Year(s) Of Engagement Activity 2018
URL https://devneuro.org/cdn/news-detail.php?NewsID=298&type=93