Periodic Reporting for period 1 - TREXSpliceosome (Investigating the structural basis of TREX function in mRNA export using cryo-electron microscopy and super-resolution fluorescence microscopy)
Reporting period: 2022-09-01 to 2024-08-31
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.
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.
In this project, we made important progress in understanding the following question: How are spliceosomes actively recycled after they hand over the mRNA to the packaging machinery? For this, we isolated spliceosomes just after release of the mRNA, and imaged them using an advanced imaging technique known as cryogenic electron microscopy. By collecting large amounts of data on state-of-the-art microscopes, we were able to obtain a detailed three dimensional structure of these late spliceosomes at a resolution of 2.6Å. Using this data, we could decipher in atomic detail how three RNA molecules and 40 individual proteins come together to form the late spliceosome. This allowed us to identify five factors that start breaking the spliceosome down into its many building blocks to initiate its recycling. Importantly, by comparing our structure to previous data, we identified that these “disassembly factors” can only bind to the spliceosome after the mRNA is released. We studied how this process works in human cells, and also in a simple model organism, the worm C. elegans. Remarkably, despite ~500 million years of evolution separating these two species, the molecular details of this complicated disassembly reaction are virtually identical. Taken together, we identified a conserved molecular quality control mechanism that ensures that only spliceosomes that have completed their task are broken down.
These findings have been presented at several international conferences (i.e the “RNA society meeting”), in a peer-reviewed scientific article (and Vorländer, Rothe, Kleifeld et al, Nature, 2024), and explained using molecular animations on social media (https://x.com/MVorlandr/status/1806220687742288205(opens in new window)) and highlighted in press releases (i.e https://www.imp.ac.at/news/article/scientists-reshape-our-understanding-of-messenger-rna(opens in new window)).
These findings have been presented at several international conferences (i.e the “RNA society meeting”), in a peer-reviewed scientific article (and Vorländer, Rothe, Kleifeld et al, Nature, 2024), and explained using molecular animations on social media (https://x.com/MVorlandr/status/1806220687742288205(opens in new window)) and highlighted in press releases (i.e https://www.imp.ac.at/news/article/scientists-reshape-our-understanding-of-messenger-rna(opens in new window)).
In this project, we addressed a long-standing question in the field of gene expression. While we knew how spliceosomes assemble on pre-mRNAs, it was unknown how they can be disassembled in a regulated way. We could further for the first time assign functions to several proteins that were known to be involved in splicing, without understanding their function on the molecular level. This projected also demonstrated how remarkably similar fundamental cellular processes are in highly divergent organisms such as nematodes and humans. The data illuminate principals of molecular quality control, ensuring faithful expression of genetic information.