Microtubules (MTs) are a crucial part of the cytoskeleton and play vital roles in cellular architecture, intracellular transport, and mitosis. Importantly, aberrations of MTs are associated with a wide range of human pathologies, and they are a critical drug target for cancer and other conditions. The dynamic nature of MTs critically depends on the availability of free tubulin subunits. Forty years ago, it was recognized that cells sense excess free tubulin and then trigger degradation of the encoding mRNAs. This process of “tubulin autoregulation” requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates mRNA decay was unknown when the project was started.
TTC5 binds at the ribosome exit tunnel and has no catalytic activity. The goal was to find the missing factors required for tubulin mRNA degradation, and to understand their molecular function. Our first discovery, using an unbiased proximity-biotinylation proteomics strategy, was that the poorly characterized protein SCAPER is a critical interactor of TTC5-bound ribosomes. We reconstituted this complex between tubulin-translating ribosomes with TTC5 and SCAPER bound, and in a collaboration with the Passmore lab (MRC-LMB), we obtained a structure that explains the selectivity of SCAPER for TTC5-bound ribosomes. Structure-guided mutants that selectively perturbed key interactions in this complex are completely deficient in tubulin mRNA decay.
SCAPER does not display nuclease activity, hence the mechanism of mRNA decay was still mysterious. Using a similar proximity-biotinylation approach, we then uncovered that SCAPER acts as an adaptor for the CCR4-NOT deadenylase complex. SCAPER directly interacts with the CNOT10/CNOT11 module to initiate tubulin mRNA decay by deadenylation. We could therefore assign molecular functions not only to SCAPER, but also to CNOT10 and CNOT11, which form a previously poorly characterized module of the CCR4-NOT complex. Importantly, I showed that disease-related SCAPER mutations abolish tubulin autoregulation and cause mitosis defects in cells (together with Dr. Ivana Gasic, Univ. of Geneva). SCAPER mutations cause a ciliopathy syndrome with neurodevelopmental defects and retinitis pigmentosa, allowing us to directly link the tubulin autoregulation pathway to human disease for the first time. This work represents the first mechanistic explanation for how recognition of the nascent protein is relayed for selective degradation of the encoding mRNA.
Our results have been published in a high-impact journal (Höpfler et al., Mol Cell, 2023, DOI: 10.1016/j.molcel.2023.05.020) and we have summarized our work and the current literature in a review article (Höpfler & Hegde, Mol Cell, 2023, DOI: 10.1016/j.molcel.2023.07.014). Furthermore, the work was presented at four major scientific conferences, was disseminated via social media networks such as Twitter/X, and was highlighted on the website of a Cambridge University College. Complementing the dissemination to academic audiences, public outreach activities of the research fellow, such as during the MRC Laboratory of Molecular Biology Open Day 2023 helped to highlight the impact of the European Union’s Horizon 2020 research and innovation programme to the general public.
