Splicing Fidelity: Enforcement, Modulation and Impairment.

Informacje na temat projektu

SPLIFEM

Identyfikator umowy o grant: 101076093

DOI

10.3030/101076093

Data podpisania przez KE 26 Kwietnia 2023

Data rozpoczęcia 1 Września 2023

Data zakończenia 31 Sierpnia 2028

Finansowanie w ramach

• European Research Council (ERC)

Koszt całkowity

€ 1 499 513,00

Wkład UE

€ 1 499 513,00

Koordynowany przez

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

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

Okres sprawozdawczy: 2023-09-01 do 2026-02-28

Podsumowanie kontekstu i ogólnych celów projektu

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.

Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty

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.

Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)

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.