Identifying the functions of a family of nuclear RNA binding proteins that switch expression between somatic and meiotic cells

Lead Research Organisation: Newcastle University
Department Name: Biosciences Institute

Abstract

PURPOSE OF RESEARCH. Vertebrate genes make extensively processed RNAs guided by RNA binding proteins that are often encoded by multigene families. However, the individual functions of proteins within these families are frequently not well understood. The purpose of this research is to provide the first global analysis of the RBMX gene family which is conserved in all mammals, and has members conserved in all vertebrates. The RBMX family of nuclear RNA binding proteins consists of RBMX, hnRNP GT and RBMY proteins, and is interesting since there are gene expression switches between somatic cells (found all over the body) and meiotic cells (that eventually give rise to sperm). Preliminary mouse knockout data indicates that these gene expression switches are critical, but why this is or what individual RBMX family proteins do is still largely unknown.

TIMELINESS AND VALUE FOR MONEY. We have already established conditions in which we can deplete the RBMX protein in somatic cells, and induce changes in RNA processing of one endogenous target transcript, which we will be able to scale up to a more global analysis. We have also made a new mouse model, from which we can delete the highly conserved Hnrnpgt gene. Although these knockout mice are otherwise healthy, Hnrnpgt deletion causes germ cells to arrest during meiosis at a crucial stage called diplotene. The combination of these two advances, RBMX knockdown and hnRNP GT deletion, provide us the opportunity to understand how the coordination of RBMX and hnRNP GT protein activity controls pathways of nuclear gene expression.

OBJECTIVES. Objective 1 will identify target genes and pathways controlled by the RBMX protein in somatic cells, and mechanisms of RBMX function. Objective 2 will search for genes and pathways regulated by hnRNP GT protein in meiosis, and seek to determine if these are the same or different to those regulated by RBMX in somatic cells. Although we don't know what pathways are disrupted without hnRNP GT protein, we can make a strong prediction from the detailed phenotype of our Hnrnpgt null mice. HnRNP GT null germ cells arrest during meiosis at diplotene, an important stage where the paired chromosomes are just starting to separate. Our preliminary data show that meiotic chromosomes are abnormally spread out in Hnrnpgt null testes, suggesting at least some of the targets of hnRNP GT will control meiotic chromosome structure. Interestingly RBMX has also been connected to chromosome structure, but the mechanism is not known. Objective 3 will take further advantage of our new mouse model to determine if hnRNP GT has important functions after meiosis, during which time RBMX is still turned off. Objective 4 will test if the RNA processing pathways we find to be controlled by mouse RBMX and hnRNP GT are conserved in humans, and mis-regulated in infertile men.

Taken together, these objectives will dissect individual functions within this highly conserved family of RNA binding proteins, and how switches in expression between individual proteins control global patterns of gene expression. In particular, we will test how the switch between RBMX and hnRNP GT expression contributes to changes in global splicing programmes.

OUTCOMES. The expected outcomes from this research will be a better understanding of how families of nuclear RNA binding proteins function to control pathways of development. Such programmes are likely important all over the body. This project will shed new light on how the RBMX proteins are important to human health. RBMX family genes are implicated in human male infertility, brain and muscle development and cancer, but still poorly understood. This would be the first comprehensive analysis of this family of RNA binding proteins. The beneficiaries of this project will be scientists interested in gene expression and development, scientists and students that we train, infertile men and members of the public with whom we engage.

Technical Summary

This project will address functions within a protein family of nuclear RNA binding proteins that switch expression between somatic body cells (most of the cells in the body) and meiotically active cells (that ultimately make the next generation).

We will decipher global mechanisms of RNA processing control by RBMX using already established protocols to deplete RBMX expression, and then RNAseq to identify direct and indirect targets. Direct RBMX targets will be distinguished using iCLIP (a powerful crosslinking approach that can reveal global endogenous RNA targets that are bound to RBMX). RBMX protein turns off in meiosis and is replaced by another family member called hnRNP GT. In our preliminary experiments we find this switch in gene expression is crucial, since mice null for the Hnrnpgt gene arrest germ cell development during meiosis at a stage called diplotene. Both wild type and Hnrnpgt null mice have similar cell type contents at developmental stages before this block (postnatal day 18). We will globally identify RNA targets of hnRNP GT protein by RNAseq, comparing day 18 mouse testes from wild type and knockout mice to identify mis-regulated RNA processing pathways. Direct hnRNP GT target RNAs will be identified using iCLIP to address patterns and mechanisms of regulation, and to what extent these are similar or overlap with RBMX. We will monitor RNA processing pathways of endogenous transcripts over germ cell development to establish whether hnRNP GT provides a direct or specialised replacement for RBMX in meiosis, and how this fits into the global transitions in RNA processing pathways occuring during germ cell development. To establish if hnRNP GT is also needed after meiosis, we will cross our floxxed mouse model with a Prm-Cre expressing mouse to inactivate hnRNP GT after diplotene. Finally we will examine RNA processing patterns in human testis RNA to see if the RNA processing events controlled by RBMX and hnRNP GT are conserved.

Planned Impact

BENEFIT TO PATIENTS WITH MALE INFERTILITY AND THEIR CLINICIANS
WHO WILL BENEFIT? Infertile men with arrested meiosis are frequently seen in clinics, but there is very little that can be done to diagnose or otherwise help them apart from Y chromosome deletion mapping.
HOW WILL PATIENTS AND CLINICIANS BENEFIT? The identification of new pathways important for completion of meiosis will potentially lead to new genetic tests, and in the long term to the development of blood-based tests that could obviate the need for testis biopsies that are expensive and potentially hazardous. The research in this grant could therefore increase efficiency within the NHS, influence medical practitioners, and be of potential application in the development of diagnostic kits. We will also interact with the Infertility Network UK, a charity that promotes the interests of people with infertility. Although not life threatening, infertility can be psychologically damaging, particularly if it is of unknown origin.

DELIVERING AND TRAINING HIGHLY SKILLED RESEARCHERS.
WHO WILL BENEFIT? Students at Newcastle University and professional scientists working on the project.
HOW WILL TRAINING BENEFIT FROM THIS RESEARCH? An important impact of our work will be in science education. Newcastle University is a research led university, and work in the lab feeds through into taught classes as well as projects carried out by undergraduate and postgraduate students. In the case of lab based projects, students get the opportunity to become directly involved for a time in research projects, and both the PI and Research Coinvestigator on this grant are involved in project student supervision. The impact in science education from this grant will be immediate (Years 1-3).This project will also enhance the professional research skills of the Research Co-investigator on this project, who will interact with local bioinformatics support for the RNAseq analysis.

BENEFIT TO SOCIETY
WHO WILL BENEFIT. We will contribute to cultural enrichment, by reaching out to local organisations interested in science and schools. We expect male infertility to be of interest to the press.
HOW WILL SOCIETY BENEFIT FROM THIS RESEARCH? We will provide talks to the general public about this project (Café Scientifique, Year 3). We will host sixth form students who are interested in a career in science or medicine, give talks at Science and Engineering weeks at local schools, and issue press releases with papers (Years 1-3 of grant).


ENHANCING THE KNOWLEDGE ECONOMY
WHO WILL BENEFIT? A longer term social and economic impact of our study will be in the continued development of the local science infrastructure. Newcastle is a post-industrial city in which the University is a major local employer, and is developing itself as a Science City in which the economy is based on a foundation of research and technology. Within the North East of England over 140,000 people are employed in biotechnology, life sciences, NHS and associated organisations. The Institute of Genetic Medicine (IGM) is part of the city centre-located International Centre for Life, which includes both aspects of Science education and start up companies, and links with local patient care under the NHS. Key to this vision is Research Excellence.
HOW WILL THE LOCAL ECONOMY BENEFIT FROM THIS RESEARCH? Local expertise in the application of post genomic technologies will make an important contribution to the local scientific infrastructure. Our high profile research also acts to bring in international students and scientists, which has a positive knock on effect on the local economy.
 
Description The hnRNPGT gene is found in all mammals, but it was not known what it is needed for. We used mouse genetics to identify the functions for hnRNP GT regulation during male germ cell development - these are the cells which give rise to sperm in the testis. We made a conditional mouse knockout, and used this to delelte the gene encoding hnRNP GT. This caused an arrest during meiosis - the specialised form of cell division that makes sperm. hNRNP GT is an RNA binding protein, and so we thought it could either control RNA synthesis or RNA processing. Most human genes are processed by RNA splicing, in which segments called exons are joined together to make functional mRNAs. We analysed patterns of gene expression to find out what was going wrong in these mice. What we found is that there are bits of RNA being inserted into the mRNAs from genes. These bits of RNA are "spliced in", since they look a bit like normal segments of mRNAs, but are normally ignored. These extra segments are called cryptic exons. As a result, we think that gene expression patterns are being poisoned during meiosis, and this is causing the cells to arrest development. This work was published last year in eLife. The official name for hnRNP GT is RBMXL2. eLife published a non-specialist "digest" at the start of this article, which gives a general reader summary of the work.

We have also identified RBMX targets and are following this up.
Exploitation Route Other researchers can download and use our RNAseq information.
Sectors Education,Healthcare

URL https://elifesciences.org/articles/39304
 
Description We have used our findings so far in the public engagement Genetics Matters event. We have also had high school students visit the lab, and visited schools as well. Our eLife paper was picked up by news agencies around the world, and highly tweeted. based on this research, I was asked to contribute to a Newsweek article, which would have been highly read around the world.
First Year Of Impact 2018
Sector Education,Healthcare
Impact Types Societal

 
Title Mouse with conditioal allele of Hnrnpgt gene 
Description We flanked the mouse Hnrnpgt gene with Cre sites. This can be used to excise this gene using Cre recombinase. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? Yes  
Impact None yet 
 
Title HITS clip map of hnRNP GT binding in the mouse testis 
Description This is a physical list of targets bound by hnRNP GT in the mouse testis. It will be released when we publish our paper. 
Type Of Material Database/Collection of data 
Year Produced 2011 
Provided To Others? No  
Impact This database is helping us interpret our data 
 
Title RNAseq dataset GSE101511 
Description RNAseq dataset deposited at GEO for 18day old testis from wild type and Hnrnpgt-/- male mice 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact None yet, although it formed the basis of our 20198 eLife paper 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101511
 
Description Collaboration with Lorna Harries, Exeter University 
Organisation University of Exeter
Department Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution We used an extensive collection of normal and prostate tumour RNA isolated by the Harries group
Collaborator Contribution This has resulted in two papers so far, and two in preparation
Impact Munkley et al, 2015, Oncotarget, and Munkley et al 2015 Oncoscience
Start Year 2014
 
Description Collaboration with University of Pennsylvania 
Organisation University of Pennsylvania
Country United States 
Sector Academic/University 
PI Contribution Generation of NGS data and validation of results
Collaborator Contribution Analysis of NGS data using cutting edge algorithm
Impact This is still in progress currently
Start Year 2015
 
Description Genetics Matters 
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 There was very good attendance for this event -108 people were registered. I discussed our BBSRC funded work at a research table, and displayed some of our data. I also showed attendees some microscope slides of human and mouse testes stained for hnRNP GT. Most people attended my table at some point, and there were both questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Interview for international magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I was interviewed by Newsweek based on the press being aware of the work we published from this grant on male infertility.
Year(s) Of Engagement Activity 2019
URL https://www.newsweek.com/picking-sex-baby-scientists-x-ychromosome-sperm-implications-1453756
 
Description Visit from schools for work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact These were very successful visits by school students who spent a week in our institute, including 1.5 days in my lab working on actual scientific projects and analysing data

We were thanked by the schools and parents, and the students were very enthusiastic
Year(s) Of Engagement Activity 2012,2013
 
Description Visiting school to talk about science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact The school were really grateful to my BBSRC funded student and Path Society student for teaching them molecular biology techniques for a day, and providing careers advice
Year(s) Of Engagement Activity 2019
URL https://twitter.com/HA_BiologyDept/status/1181283062086275072
 
Description lab twitter account 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We report events in the lab - talks, papers, visits
Year(s) Of Engagement Activity 2018,2019,2020
URL https://twitter.com/TheElliottLab
 
Description press release associated with paper 
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 issued a press release to go along with our 2019 eLife paper. This was picked up by international news agencies (online based) and a local newspaper. For this press release we interacted with an infertility charity.
Year(s) Of Engagement Activity 2019
URL https://www.sciencedaily.com/releases/2019/01/190123191631.htm
 
Description visit from high school students for work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 3 students did their work experience placement with us. They reported that it helped them make decisions about careers and university courses. We designed the placements so that the students got a broad range of experience, sharing between participating labs. The school and parents were very grateful for the work we did with them.
Year(s) Of Engagement Activity 2019,2020
 
Description visiting school to talk about research and careers in science 
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 The school were very grateful for our efforts in organising this event, which was attended by the A level biology class

"Thank you so much for your presentation this afternoon. It was really good for the students (and me) to hear about how the science they learn in the lab is relevant in the real world and I also very much enjoyed the content. It was well attended despite the ski trip meting and I think that reflects the interest our pupils have for science, something I'm keen to promote. Alice and Chile were a great addition and spoke passionately about their role as researcher and they were excellent role models for the girls who attended."
Year(s) Of Engagement Activity 2019
URL https://twitter.com/TheElliottLab/status/1201910825897938944