Michrowska, Aleksandra; (2024) Control of MRTF-SRF pathway activity by G-actin. Doctoral thesis (Ph.D), UCL (University College London).

Text Thesis_Final_Corrected_02.12.2024.pdf - Accepted Version Access restricted to UCL open access staff until 1 January 2026. Download (13MB)

Abstract

The Myocardin-Related Transcription Factors (MRTF-A and MRTF-B) are G-actin-binding proteins belonging to the RPEL protein family. They regulate the activity of Serum Response Factor (SRF) via activation of the Rho-actin pathway. MRTFs act as G-actin sensors in the cell, with signal-induced depletion of the monomeric actin pool inducing MRTF nuclear accumulation and transcription initiation. MRTF senses G-actin through its N-terminal RPEL domain, which contains five binding sites for actin, located on three RPEL motifs and two intervening spacers. MRTF binds to SRF through a heptapeptide sequence located downstream of the RPEL domain. Actin regulates MRTF at two levels: it prevents MRTF nuclear localization and inhibits SRF-DNA binding in the nucleus. Since the actin- and SRF-binding sites on MRTF are separate, it presented with a question of how actin could inhibit SRF-DNA binding. We successfully reconstituted this inhibitory role of actin in vitro, showing that actin binding to MRTF prevents the formation of the MRTF-SRF complex. RPEL3 was crucial for this inhibition, and further analysis revealed that sequences downstream of the RPEL domain stabilize its interaction with actin. Using AF2-Multimer in silico modelling, we identified a novel actin-binding region on MRTF—the Q-box. This finding was confirmed by HDX-MS analysis, which showed that recruitment of the Q-box to a composite site on RPEL3-actin was dependent on the integrity of RPEL3. Disruption of the interaction between RPEL3 and the Q-box lowered the overall affinity of MRTF for actin. As the Q-box sequences are proximal to the SRF-binding site, our work suggests mutual exclusivity between MRTF-SRF binding and MRTF-actin binding. This will be further validated in vivo. We propose a model in which actin binds to a composite high-affinity site comprising RPEL3 and the Q-box, nucleating the assembly of a higher-order pentavalent RPEL-actin complex.

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