TREXSpliceosome · Investigating the structural basis of TREX function in mRNA export using cryo-electron microscopy and super-resolution fluorescence microscopy
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2022-09-01 → 2024-08-31
- EU contribution
- €174,167
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Investigating the structural basis of TREX function in mRNA export using cryo-electron microscopy and super-resolution fluorescence microscopy
Genetic information is stored in DNA. Genes generally contain the instructions on how to produce one specific protein. When a gene is activated, the cellular machinery produces a copy of this gene in the form of pre-messenger RNA (pre-mRNA) in a process called transcription. Pre-mRNA is an immature molecule that needs to be processed in order to form a functional mRNA that contains useful instructions. This processing includes two important events: pre-mRNA splicing, in which non-coding information is removed from the pre-mRNA, and mRNA packaging, in which the mature mRNA is organized into a specific three-dimensional shape. Only when both these steps, splicing and packaging, are completed successfully can the mRNA be exported from the cell nucleus into the cytoplasm, where it can be used to make new proteins. The aim of this project was to better understand the molecular machines involved in pre-mRNA splicing and packaging, and how they work together. Specifically, it was unclear how the splicing machinery, (called the spliceosome), hands over the mature mRNA to the packaging machinery (also known as TREX, for transcription-export complex). After handover, the spliceosome also needs to be actively disassembled, or “recycled”, in order to regenerate spliceosome components. To tackle these questions, we set out to use genome engineering and advanced electron microscopy methods (known as cryo-EM) to isolate spliceosomes, mRNAs, and TREX complexes from human cells and obtain detailed images of these complex molecular machines at different stages of their life cycle. This would allow us to understand how these machineries coordinate their activities and how it is ensured that spliceosomes are only disassembled once they have executed their task.
Data: CORDIS, © European Union
Project objective
Eukaryotic mRNA is transcribed, capped, spliced and poly-adenylated in the nucleus, but translated in the cytoplasm. Nuclear mRNA export is a key step in gene expression which is still poorly understood in mechanistic detail, but the multi-subunit transcription and export (TREX) complex plays a central role in the process. TREX bridges transcription, RNA processing and packaging to facilitate mRNA export licensing. Understanding TREX function is relevant to human health, as TREX protects against transcription-associated genome instability, is required for development, and has been linked to human disease, including several types of cancer. Despite its importance, many questions about TREX function remain, in particular, how is TREX recruited to mRNA and how is the export factor NXF1-NXT1 deposited on mature mRNA? Here, I propose to study the mechanistic basis of TREX recruitment and function using structural biology and imaging approaches. The host laboratory recently purified an endogenous multi-megadalton spliceosome–TREX complex, whose three-dimensional cryo-EM structure I aim to determine. This will reveal how TREX is recruited to maturing mRNA to read out and relay the presence of activating and repressive marks to, ultimately, decide whether or not to export the mRNA. In close collaboration with the Balzarotti lab (IMP), I will use super-resolution microscopy (MINFLUX) to visualize post-recruitment functions of TREX in situ and test models of how deposition of the mRNA export factor NXF1-NXT is linked to export competence. Taken together, the proposed work will significantly enhance our understanding of TREX as a chaperone of nuclear mRNA processing and export.
Original text from CORDIS.
Participants
- FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH · WienCoordinatorAustria
Links
Data: CORDIS, © European Union