Periodic Reporting for period 1 - mRNA-DEG-RIBOSOME (Understanding molecular principles of regulated mRNA degradation on translating ribosomes)
Reporting period: 2021-10-01 to 2023-09-30
Cellular function fundamentally hinges on maintaining defined protein levels, a process tightly regulated by messenger RNA (mRNA) synthesis and decay. Excess protein production is not only costly to cells but can also be dangerous, because certain surplus proteins tend to misfold, aggregate, and cause disease. Furthermore, novel RNA-based therapeutics such as mRNA vaccines that helped curb the global COVID-19 pandemic (Nobel Prize in Physiology or Medicine 2023) require a deep mechanistic understanding of cellular mRNA regulation to improve innovative drug design.
Cells tightly regulate mRNA processing, localisation and stability to ensure accurate gene expression in diverse cellular states and conditions. Most of these mRNA regulation steps have traditionally been thought to primarily happen before translation. However, recent discoveries highlight the role of nascent polypeptides on translating ribosomes in the active regulation of mRNAs. As the nascent protein emerges from the ribosome, it marks the identity of the encoding mRNA by its unique amino acid sequence. This allows cotranslational engagement by specific polypeptide recognition factors on the ribosome, providing them access to the associated mRNA. A striking example for such regulation is the negative feedback loop in the expression of tubulins known as “tubulin autoregulation”. In this case, tubulin mRNA is degraded cotranslationally when cells sense excess free tubulin, which is critical during mitosis to ensure faithful chromosome segregation. Recently, the first specific factor in this pathway has been identified: TTC5 recognises the N-terminus of nascent tubulin emerging from the translating ribosome and triggers degradation of the associated mRNA. However, the events leading to tubulin mRNA decay downstream of TTC5 remained elusive before this project.
In mRNA-DEG-RIBOSOME, the tubulin autoregulation pathway was used as a model system to study how the nascent protein chain on translating ribosomes directs mRNA degradation. Specifically, the aims were to: (1) identify factors acting downstream of TTC5 required for tubulin mRNA degradation using cutting-edge mass spectrometry and genetic screening methods; (2) understand the mechanistic role of each factor in TTC5-directed mRNA degradation by using a powerful in vitro reconstitution approach; and (3) expand the understanding of nascent chain-dependent mRNA degradation by investigating related feedback systems. mRNA-DEG-RIBOSOME aimed to establish a conceptual framework for how cells have evolved to exploit nascent polypeptide recognition to direct mRNA fate.
Cells tightly regulate mRNA processing, localisation and stability to ensure accurate gene expression in diverse cellular states and conditions. Most of these mRNA regulation steps have traditionally been thought to primarily happen before translation. However, recent discoveries highlight the role of nascent polypeptides on translating ribosomes in the active regulation of mRNAs. As the nascent protein emerges from the ribosome, it marks the identity of the encoding mRNA by its unique amino acid sequence. This allows cotranslational engagement by specific polypeptide recognition factors on the ribosome, providing them access to the associated mRNA. A striking example for such regulation is the negative feedback loop in the expression of tubulins known as “tubulin autoregulation”. In this case, tubulin mRNA is degraded cotranslationally when cells sense excess free tubulin, which is critical during mitosis to ensure faithful chromosome segregation. Recently, the first specific factor in this pathway has been identified: TTC5 recognises the N-terminus of nascent tubulin emerging from the translating ribosome and triggers degradation of the associated mRNA. However, the events leading to tubulin mRNA decay downstream of TTC5 remained elusive before this project.
In mRNA-DEG-RIBOSOME, the tubulin autoregulation pathway was used as a model system to study how the nascent protein chain on translating ribosomes directs mRNA degradation. Specifically, the aims were to: (1) identify factors acting downstream of TTC5 required for tubulin mRNA degradation using cutting-edge mass spectrometry and genetic screening methods; (2) understand the mechanistic role of each factor in TTC5-directed mRNA degradation by using a powerful in vitro reconstitution approach; and (3) expand the understanding of nascent chain-dependent mRNA degradation by investigating related feedback systems. mRNA-DEG-RIBOSOME aimed to establish a conceptual framework for how cells have evolved to exploit nascent polypeptide recognition to direct mRNA fate.
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.
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.
In conclusion, the mRNA-DEG-RIBOSOME project could solve a 40-year-old puzzle of how tubulin mRNA is degraded when there is too much tubulin protein in the cell. This work represents the first mechanistic explanation for how recognition of the nascent protein is relayed for selective degradation of the encoding mRNA. Therefore, we provide a conceptual framework for future work on translation-coupled mRNA regulation.
Understanding the molecular mechanisms of cellular mRNA regulation is a crucial prerequisite for the design of novel RNA-based therapeutics, such as mRNA vaccines. Thus, our work might in the long run lead to improved drug design. Furthermore, as a long-term perspective, it might be possible to re-purpose the mechanisms we discovered for highly targeted degradation of cellular mRNA substrates. This is particularly relevant for diseases where individual genes are overexpressed with detrimental effects to human health, such as oncogenes in cancer, or aggregation-prone proteins that cause neurodegenerative diseases such as Alzheimer’s or Huntington’s disease.
Understanding the molecular mechanisms of cellular mRNA regulation is a crucial prerequisite for the design of novel RNA-based therapeutics, such as mRNA vaccines. Thus, our work might in the long run lead to improved drug design. Furthermore, as a long-term perspective, it might be possible to re-purpose the mechanisms we discovered for highly targeted degradation of cellular mRNA substrates. This is particularly relevant for diseases where individual genes are overexpressed with detrimental effects to human health, such as oncogenes in cancer, or aggregation-prone proteins that cause neurodegenerative diseases such as Alzheimer’s or Huntington’s disease.