Biology subjects

Richards, M. W.

Publications and source records attributed to Richards, M. W..

3 recordsLinked to original sources

bioRxiv2025

The MYCN/Aurora-A complex is a cyclin activating kinase for CDK12

Deregulated MYCN is a driver of aggressive pediatric and adult neuroendocrine tumors, but critical oncogenic processes downstream of MYCN remain poorly defined. In neuroblastoma, MYCN interacts with and activates the Aurora-A kinase. Here we show that Aurora-A is a CDK-activating kinase for CDK12 by phosphorylating T893 in the T-loop, thereby enhancing its kinase activity. Aurora-A-dependent activation of CDK12 controls phosphorylation of T4 of RNA polymerase and recruits transcription termination complexes, thereby preventing transcription-replication conflicts. Enhanced crosslinking and immunoprecipitation sequencing reveals that Aurora-A associates with splice sites on nascent RNA. RNA-bound Aurora-A is catalytically inactive. MYCN competes with RNA for binding to Aurora-A and displaces Aurora-A from RNA in cells, promoting its CDK12 kinase activity. Combining Aurora-A and CDK12 inhibition potently suppresses the growth of MYCN-amplified neuroblastoma cells and patient-derived xenografts. Our data demonstrate that an Aurora-A/CDK12-dependent transcription termination pathway is a critical and targetable dependency of MYCN-driven tumors.

cancer biology↗

bioRxiv2024

Structural characterization and inhibition of the interaction between ch-TOG and TACC3

The mitotic spindle is a bipolar array of microtubules, radiating from the poles which each contain a centrosome, embedded in pericentriolar material. Two proteins, ch-TOG and TACC3, have multiple functions at the mitotic spindle due to operating alone, together or in complex with other proteins. To distinguish these activities, we need new molecular tools to dissect their function. Here, we present the structure of the -helical bundle domain of ch-TOG that mediates its interaction with TACC3 and a structural model describing the interaction, supported by biophysical and biochemical data. We have isolated Affimer tools to precisely target the ch-TOG-binding site on TACC3 in live cells, which displace ch-TOG without affecting the spindle localization of other protein complex components. Inhibition of the TACC3-ch-TOG interaction led unexpectedly to fragmentation of the pericentriolar material in metaphase cells following the formation of a bipolar spindle and delayed mitotic progression; uncovering a novel role of TACC3-ch-TOG in maintaining pericentriolar material integrity during mitosis to ensure timely cell division.

cell biology↗

bioRxiv2024

Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR

The myc family of proteins (c-, N- and L-myc) are transcription factors (TFs) responsible for maintaining the proliferative program in cells. They consist of a C-terminal domain that mediates heterodimerisation with Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. Structural characterization of the TAD of N-myc, is very limited, with the exception of a crystal structure of Aurora-A bound to a helical region of N-myc. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment enabled by a truncation approach. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: one region within Trp77-Glu86 and the second between Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with another known interaction site (myc box I), have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and showed no significant structural changes through the rest of the TAD. When doubly phosphorylated on residues Ser62 and Thr58, N-myc formed a robust interaction with the Fbxw7-Skp1 complex. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

biophysics↗