Decoding Hox specificity from mRNA processing networks

Project Information

RNA-NetHOX

Grant agreement ID: 101024467

DOI

10.3030/101024467

Project closed

EC signature date 6 April 2021

Start date 1 January 2022

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 184 707,84

EU contribution

€ 116 953,92

Coordinated by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - RNA-NetHOX (Decoding Hox specificity from mRNA processing networks)

Reporting period: 2022-01-01 to 2023-12-31

Summary of the context and overall objectives of the project

Eukaryotic gene expression is remarkable: from limited genetic material, it creates various proteomes and cell types. To do so, genes are transcribed into pre-mRNAs that undergo processing including splicing, the mechanism of exon/intron retention or excision, to produce mature mRNAs. A single pre-mRNA can be spliced in different ways thereby diversifying transcript isoforms and proteins. This mechanism termed alternative splicing contributes to the diversity of cell and tissue identities in complex organisms. Conversely, aberrant splicing leads to severe pathologies such as neuromuscular disorders and cancers. However, realising how such transcriptional and splicing programs are coordinated is still a challenge in Biology.
Transcription factors (TFs) are the key players in gene expression by triggering precise spatial-temporal transcriptional programs. If most TFs act at the DNA regulatory layer, some TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic clues underlying TF function in alternative splicing remain elusive. Solving this issue will provide unique entry points to understand the mechanisms orchestrating cell and tissue diversity in animals and their aberrant regulation in diseases.

To address this central issue in gene regulation, we use the Hox TF as a model based on our recently published work. Specifically, the research objectives were the following:
-Decipher how interactions with splicing factors could impact Hox tissue-specific functions
-Enlarge the cooperative role of mRNA processing regulators and HOX proteins

As outcomes of the project, we have characterised the interaction between the Hox TF Ultrabithorax (Ubx) and splicing factors in the mesoderm of Drosophila embryos. We have generated a genetic toolkit for studying Ubx splicing function and the toolkit for assessing the Ubx/splicing factors function in vivo is in progress. We have determined the conservation of HOX/splicing factors interaction in humans and established a protocol to identify the interactome of these complexes. Capturing these complexes and characterising their function as outlook will contribute to a better understanding of the mechanisms governing cell and tissue diversity and their deregulation in diseases.

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

Using molecular biology and extended genetics in vivo, we characterised the interaction between the transcription factor Ubx and two splicing factors, in the mesoderm of Drosophila embryos. We have further dissected one of the mechanisms by which Ubx regulates splicing in Drosophila cells. As a milestone, we have established the first Ubx genetic toolkit to distinguish its transcription and splicing function. Moreover, we have validated the interaction of splicing factors with several human HOX proteins by bi-molecular fluorescent complementation assay and designed a proteomic-based method to capture the interactome of dimeric complexes in human cells that we will use in future in the physiological context of stem cell differentiation.
The results have been disseminated at international conferences in RNA biology and Drosophila meetings and public outreach at Science Day. The results have been exploited through an open-access review publication (DOI:10.1002/wrna.1752) and will be further exploited in 2024 with a manuscript in preparation.

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)

At the training level, the fellow enriched her skills in coordinating research projects and in the supervision of an engineer and students. The fellow has also extended her scientific networks by collaborating with experts in organoid and structural biology. Importantly, the mentoring support at the host institute has been a valuable resource for the fellow’s career development, offering guidance for developing confidence and leadership within the scientific community.
On the scientific aspect, the fellow has contributed to the scientific community by co-organizing a scientific webinar and a mentoring program for PhD students and postdocs. The fellow has also participated in various public outreaches including workshops presenting Drosophila as a biological model.
Beyond personal development, the achievement of the research program provides a unique contribution to the Hox field and more globally on gene regulation by shedding light on a new layer of TF specificity at the RNA regulatory layer. These results and subsequent outlooks will extend our understanding of the molecular mechanism orchestrating cell diversity in animals and their deregulation in diseases.

multilayered function of TFs in transcription and splicing