The role of RNA export factors in tissue specific gene expression

Individual cells in multicellular organisms are have distinct shapes and characteristics, each specialised for a specific function. A major aim in understanding organism function is to understand how cells become specialised (differentiated). Every cell in a multicellular organism carries the same genes. Cells differentiate by expressing genes for proteins relevant to their final function, and repressing genes whose protein products are not needed (or are even detrimental) for the final function. This project is aimed at increasing understanding of the fundamental biological processes that control gene expression in differentiating cells. We focus on sperm production, since sperm are extremely specialised, and their formation depends on expression of a very large number of genes not used in any other process. We use the fruit fly, Drosophila melanogaster, as a model system as it is highly amenable to a genetic and molecular analysis. Since these processes fundamental to all multi-cellular organisms, we expect our results to have broad implications, eg in understanding human development and disease.
Expression of genes requires copying (transcribing) information from DNA, in the cell nucleus, into an RNA molecule that is then transported to the cytoplasm and translated into protein. Gene expression is well known to be regulated at both transcriptional and translational levels. RNA processing in the nucleus, and export from the nucleus, also present control points. The mechanism by which RNAs get exported from the nucleus to the cytoplasm is well characterised, and relies on a set of proteins that are conserved from yeasts to humans.
Regulation of gene expression at the transcriptional level relies on the DNA of specific regions near genes "promoters" having sequences that are bound by sequence-specific DNA binding proteins (transcription factors). Binding of these transcription factors to gene promoters then recruits an enzyme complex (RNA polymerase) that actually transcribes the DNA sequence to give an RNA transcript. Usually this contains intervening sequences (introns) that get spliced out to make the final mRNA product. The process of transcription, and particularly splicing, promotes binding of the RNA export proteins to the mRNA. RNAs that are not appropriately processed are degraded.
In our recent research we discovered that a component of the RNA export pathway is required for expression of testis-specific transcripts, but not most other transcripts, in sperm development. We have shown that this is because the RNA export factors, which are understood to work downstream of the transcription factors in regulation of gene expression, actually also feed back to promote activity of a specific transcription factor in testes. Without this feedback the genes required specifically to make sperm are not expressed, and the flies are sterile.
Our project aim is to understand the mechanism of this feedback. Is the whole export pathway involved, or only specific components? Do the export factors directly bind to the transcription factors? Do the export factors promote binding of the transcription factors to the DNA, or do they promote the transcription factors activity in recruiting the RNA polymerase? Does the transcription factor help protect the testis-specific RNAs from degradation?
We also found that reducing activity of the RNA export makes the females sterile, and causes many individual animals to die as pupae. These effects are very specific, and don't appear to be due to a general failure in RNA export, but appear, like the situation in testes, to be caused by defects in specific transcription programmes. We will investigate the generality of our findings on the link between transcription and RNA export by characterising the gene expression defects in these other developmental contexts. We hope to identify the relevant transcription factor, and develop mechanistic insights using our findings from testes as a guide.

The well conserved canonical RNA nuclear export pathway exports most mRNAs from the nucleus to the cytoplasm. Most research on this pathway has either been in yeast or tissue culture cells where reduction in export pathway function led to transcripts accumulating in the nucleus. We recently discovered that the RNA export pathway also regulates tissue-specific transcription in Drosophila. Specifically, we identified a reduction in transcription of testis-specific mRNA in a hypomorphic allele of the export factor Nxt1. Intronless genes are particularly affected, and we discovered that addition of an intron to a reporter transcript increased its expression in the Nxt1 mutant. We propose that Nxt1's effect on transcription is mediated via the RNA via a feed back mechanism. Most excitingly we also discovered that the genes that require Nxt1 for expression also depend on a specific transcription complex, tMAC:- tMAC-dependent promoters impose Nxt1 dependence on reporter transcripts.
Aim 1 - the mechanism in testis: We will test whether Nxt1's role in transcription is dependent on its role in RNA export. Preliminary experiments indicate that knock down of other export factors gives the same phenotype as Nxt1 mutants. We will examine if Nxt1 promotes tMAC loading onto promoters, tMAC spreading to nearby chromatin, or loading and activation of RNA polymerase II, and systematically test for direct interactions between tMAC subunits and export factors. Unexported RNA is typically degraded via the exosome, so we will test whether tMAC and Nxt1 protect testis transcripts from the exosome. We will determine how the interaction between tMAC and the export pathway ensures high expression of intron-less transcripts in testes.
Aim 2 - other developmental contexts: Nxt1 mutants are also female sterile and semi-lethal. By detailed analysis of these defects we will determine if the export pathway's role in tissue specific gene expression extends to other developmental contexts.

This basic research is unlikely to lead to directly commercialisable results, and thus the major impact is in increasing our understanding of the basic biology of tissue specific gene expression. However, we will endeavor to ensure that the results we generate, and the methods we develop, will be disseminated widely, so the any unforeseen broader benefits can be identified and acted on.

We routinely work with very small biological samples, and thus our work impacts on the development of commercial reagents suitable for this type of research. For example have worked with molecular biology suppliers to assay their products for robustness when dealing with small samples, and found dramatic differences between apparently similar products. Our feedback of these results to the companies could lead to them developing better products for this market in the future. This would obviously benefit the suppliers, but will also improve the quality of research conduced in other laboratories. An example of this will be our use of the EU-Click-iT system for nascent RNA capture. This kit is typically used for tissue culture cells, where cell numbers are not dramatically limiting. We will optimize its use for much smaller numbers of cells, thus broadening the potential for this highly exciting technology.

HW-C has a long-standing collaboration with a UK-based SME, Oxitec. Oxitec's business is to use genetic modification to create strains of insects with engineered dominant sterility or lethality to use in population control programmes. Our existing collaboration relates primarily to developing expression systems for testes, to allow specific, controllable, activation of ectopic expression in the male germline. Our research in this project could feed into the collaboration, specifically in determining the best construct designs. For example, to date all the constructs used by Oxitec have an intron, as this helps gene expression in somatic tissues, however our findings in testes suggest that use of a tMAC-responsive promoter, and no intron, might give higher expression and better specificity. The research in this grant will help in designing the optimal construct structure. Oxitec are also interested female germline expression and our identification of the transcription factors for this system will assist in defining evolutionarily conserved promoters for development work.

The other principal beneficiaries of the research will be the two staff members. Dr Caporilli has been working part time on this project for 18 months and the grant will allow her to devote her full research effort to this area. She has had considerable input into the experimental design, and has developed excellent Drosophila genetic, cell biology and molecular biology skills. This work will extend her skill set, particularly into highly advanced molecular biology, and will set her up for a future career in molecular developmental biology. She will present the work at conferences and also in "Science Café", a venue where researchers can communicate their work directly to the public. These activities will enhance her communication skills. Dr Caporilli will also be involved in the management of the more junior staff member, so this will develop her management skills. The second staff member will be an inexperienced research assistant (to be appointed), so this person will learn state of the art techniques and will develop their skill set to continue in research (eg to do a PhD). The molecular biology skills will be transferable if this person decides to pursue a career in industry, and the communication skills developed from conference attendance, along with time management, project management, data recording etc will be of benefit in any future career.