RNA Epigenetics

Our lab investigates the general principles of gene regulation, with a focus on the coupling between transcription, RNA modifications and alternative splicing during the development of complex organisms.

Pre-mRNA splicing is crucial for the regulation of gene expression and generation of protein diversity. Most human genes undergo alternative splicing, and deregulation of this process is associated with a large number of disorders including cancer. Although alternative splicing was discovered in the 1970s, the precision and complexity of intron removal is still not fully understood. This complexity partly results from the fact that splicing is not an isolated event but occurs co-transcriptionally. The splicing machinery can therefore receive input from the transcription machinery, chromatin structure and RNA modifications, which affects the dynamics and outcome of splicing events.

Using molecular biology and classical genetics approaches combined with high throughput techniques and computational tools, our lab investigates the contribution of these processes to the regulation of alternative splicing during development. 

Figure 1. An integrated model for the regulation of pre-mRNA splicing. A combination of parameters determines the inclusion or exclusion of exons in the mature transcript. RNA features such as cis-acting elements together with transcriptional and chromatin properties modulate the recruitment of splicing factors to the pre-mRNA.

 

Interplay between transcription elongation and alternative splicing during development

Although splicing can be reproduced in vitro using an RNA template and nuclear extracts, it is now clear that most splicing events in vivo are initiated co-transcriptionally and that introns are removed while the nascent transcript is still tethered to the DNA by RNA Polymerase II (Pol II). This temporal overlap is important for the coupling between the two processes (Fig. 1). The best-documented example is the requirement for an intact RNA Pol II in pre-mRNA splicing. Through the C-terminal repeat domain (CTD) of its large subunit, RNA Pol II can recruit a wide range of proteins, including splicing factors, to nascent transcripts, thereby influencing removal of their introns. Pol II can also influence splicing via a second mechanism, referred to as kinetic coupling. According to the kinetic coupling model, modulation of transcriptional elongation rates can influence whether or not exons containing weak splice sites are recognised by the splicing machinery. Consistent with this model, accumulating evidence indicate that chromatin compaction can modulate transcription kinetics and ultimately impact pre-mRNA splicing. While these results demonstrate that transcription elongation must be tightly controlled, it remains unclear how this process is regulated during development. Our research addresses the following questions using cell culture systems and Drosophila melanogaster as a model organism:

  • What is the impact of transcription elongation on alternative splicing during development?
  • How is the transcription elongation rate regulated during development, in different tissues?
  • What is the influence of chromatin regulators on transcription elongation?
  • How does the splicing machinery feedback on the transcription elongation rate?

Coupling between RNA modifications and alternative splicing

Another layer of epigenetic regulation of gene expression is provided by RNA modifications. More than 100 RNA modifications have been discovered so far, but the molecular and physiological functions for most of them remain unknown. The most common modification of mRNA in mammals is the N6-methyladenosine RNA (m6A). m6A was recently shown to play crucial roles in several physiological processes, including embryonic cell differentiation, metabolism and the circadian clock. Importantly, m6A was also recently demonstrated to regulate alternative splicing, although the precise role of this modification in this process remains unclear. To obtain more insights, our lab has started to characterise m6A in Drosophila melanogaster and identified several critical players involved in its biogenesis and function (Fig. 2).

Figure 2: Characterisation of m6A players in Drosophila melanogaster. A complex of three proteins, Ime4, dMettl14 and Fl(2)d convert adenosine into N6-methyladenosine. Additional components may be involved to provide the functional specificity to the complex. m6A is recognised by members of the YTH family proteins, which mediate m6A functions during development. No demethylase has yet been identified in Drosophila.

We are currently addressing the following questions on this topic:

  • How does m6A impact alternative splicing?
  • What are the physiological functions of m6A in Drosophila?
  • How are m6A levels regulated during development?
  • What are the main players involved (writers, readers and erasers)?

Jean-Yves is an Associate Member of the EpiGeneSys network of scientists.

Interested PhD students, postdocs or Master's students are encouraged to apply.