Splicing Fidelity: Enforcement, Modulation and Impairment.

Project Information

SPLIFEM

Grant agreement ID: 101076093

DOI

10.3030/101076093

EC signature date 26 April 2023

Start date 1 September 2023

End date 31 August 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 513,00

EU contribution

€ 1 499 513,00

Coordinated by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - SPLIFEM (Splicing Fidelity: Enforcement, Modulation and Impairment.)

Reporting period: 2023-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

Pre-mRNA splicing is a key molecular mechanism in eukaryotic gene expression during which non-coding "introns" are excised from the coding sequences of messenger RNAs. Given the homogeneous nature of RNA, composed of just four nucleotides, determining which sequences should be removed is not a trivial task. This process is carried out by a complex molecular machine known as the spliceosome. To make its task even more challenging, the spliceosome must choose between alternative sequences and select the correct ones in a cell-type-specific manner or in response to various stimuli.What are the molecular mechanisms used by the spliceosome to ensure it selects the correct sequences to splice out? How is fidelity ensured or modulated during coupling other levels of gene expression? How can this regulation be disrupted in pathological conditions?
This project aims to explore different aspects of the regulation of splicing fidelity in humans. First, thanks to a combination of recumbent production of human splicing factors and in vitro reconstitution from human cell extracts we aim at capturing the human spliceosome at different stages of its activity, when it specifically recognize its target sequence in normal or pathological conditions. Second, we use biochemistry, proteomics and structural biology to precisely characterize the composition and architecture of the purified complexes.
These information will contribute to laying the molecular fundations to address one of the most mysterious in gene expression: how are introns chosen within pre-mRNA without mistake? Moreover this will provide a molecular framework to understand numerous human neurodevelopemental disorders.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our work has primarily focused on developing a comprehensive toolbox to reproduce, in vitro, some of the most complex transitions that occur during the splicing cycle, with the aim of uncovering the molecular mechanisms underlying fidelity checkpoints. We have made significant progress by producing and assembling many of the reagents required to reconstitute one of the most intricate remodeling steps of the spliceosome directly in the test tube. These reagents were challenging to obtain and originate from various sources, including human cell extracts and recombinant expression in E. coli and S. cerevisiae.
In parallel, we have established an efficient pipeline for the biochemical, proteomic, and structural characterization of the human spliceosome. This effort has led to a collaborative preprint on crosslinking mass spectrometry (crosslink/MS) with our collaborator, which is currently under revision.
Importantly, we have also collaborated with geneticists and clinicians to model the effects of recently identified mutations associated with neurodevelopmental disorders. We proposed that certain mutations in U4 snRNA disrupt the interaction network responsible for the accurate recognition of the 5' end of the intron. This multidisciplinary study was recently published in Nature Genetics. A follow-up study is currently submitted, in which we investigate the potential impact of mutations in U2 snRNA.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Part of our work has focused on recently identified mutations in components of the spliceosome. In collaboration with human geneticists and clinicians across Europe, we have investigated the molecular consequences of these mutations. With support from the ERC, my group proposed a mechanism for the associated pathologies, drawing on published structural and biochemical data. Ongoing work in the group aims to deepen our understanding of the loss of splicing fidelity caused by these mutations and, ultimately, to identify therapeutic strategies to correct them.
This research has also led to my involvement in the scientific advisory board for families affected by ReNU syndrome.