Get started
Work

CARM1 perturbe les interactions entre ALIX et ses partenaires en méthylant ses arginines et favorise la cytokinèse

Year: 2024
Type: dissertation
Abstract: Arginine methylation is a post-translational modification catalyzed by the 9 members of the protein arginine methyltransferase family (PRMT1-9). By adding methyl marks on arginines, PRMTs regulate their substrates' ability to bind to other proteins or nucleic acids, thereby modulating various biological processes such as gene expression, RNA metabolism or DNA damage response. Contrary to the other PRMTs, PRMT4, also known as CARM1 (Co-activator Associated aRginine Methyltransferase 1), specifically methylates arginines within proline-rich motifs. CARM1 is over-expressed in breast tumors compared to normal breast tissue and is localized both in the nucleus and in the cytoplasm of tumors cells. While the nuclear substrates and functions of CARM1 have been well described and have underscored its critical role in the nucleus, its cytoplasmic functions remain largely uncharacterized. Therefore, we investigated the CARM1 interactome by immunoprecipitating CARM1 from several breast cancer cell and HeLa cell lysates followed by mass-spectrometry analysis, and retrieved several proteins involved in vesicular trafficking, including endocytosis, vesicle transport and sorting. We showed that CARM1 interacts with two cytoplasmic proteins: ArfGAP3 (ADP-ribosylation factor GTPase-activating protein 3), a crucial modulator of vesicular trafficking, and ALIX (ALG-2 interacting protein X), an accessory protein of the Endosomal Sorting Complexes Required for Transport (ESCRT) that is involved in membrane remodeling processes such as virus budding, multivesicular body formation and cytokinesis. We demonstrated that the catalytic domain of CARM1 binds to the C-terminus proline-rich domain (PRD) of ALIX and identified the key residues involved in the interaction by cryogenic electron microscopy. We showed that CARM1 methylates two arginine residues in ALIX PRD in vitro and in cells, and we explored the functional consequence of ALIX methylation in its cellular functions. Cytokinesis is the final step of cell division leading to the physical separation of the daughter cells. During this process, the dividing cells are connected by a thin intercellular bridge, within which a platform called “midbody” facilitates the recruitment of proteins required for the abscission, including ALIX and the ESCRT machinery. Several proteomic analyses of midbody remnants, released in the extracellular medium following abscission, identified CARM1 in this structure, suggesting that CARM1 may regulate the last step of cell division. We demonstrated that CARM1 was essential during cytokinesis, as its depletion delayed the severing of microtubules in the intercellular bridge - a critical step prior to abscission - and induced the formation of multinucleated cells, mirroring the effects observed upon ALIX depletion. Mechanistically, we showed by a peptide pulldown approach that ALIX methylation impaired its interaction with several partners involved in cytokinesis, including CD2AP, CIN85, capping proteins and endophilin-A2. Further, a mutant of ALIX that is unable to bind these proteins promoted the formation of multinucleated cells, highlighting the requirement for ALIX to bind to these partners, for proper cytokinesis. Additionally, CARM1 regulated the transcription of genes coding for proteins involved in cytokinesis, suggesting that CARM1 could control the last step of cell division through several molecular mechanisms. Altogether, this study uncovered new cytoplasmic functions of CARM1 in vesicular trafficking and membrane remodeling processes and unveiled a novel post-translational modification controlling cytokinesis. This work also opens new areas of research regarding the relevance of CARM1-mediated ALIX methylation in other ALIX-dependent cellular functions such as receptor recycling, membrane repair, virus budding, and exosome biogenesis.
Language: English
Open Access status: green
OpenAlex ID: W7055103222
Cites:
Cited by:
Related to: