Periodic Reporting for period 1 - GOVERNA (Understanding the molecular principles governing mRNP architecture)
Berichtszeitraum: 2022-10-01 bis 2025-03-31
When gene expression is active in eukaryotic cells, the code embedded in DNA is transcribed into messenger RNA (mRNA). The promineant task of this courier is to get the code to the correct location within the cell where it will be translated into a protein. Both internal and external factors make this mission a tumultuous adventure: mRNA can be quite long and prone to folding on itself to form various structures, while the cell is rife with hazards in the form of enzymes that degrade RNA. Messenger ribonucleoprotein particles (mRNPs) are crucial assemblies in eukaryotic gene expression—perhaps, best likened to bodyguards of mRNA—as they are responsible for packaging, protecting, regulating and transferring mRNAs as they travel through the cell from transcription to translation. Despite their importance, these assemblies of mRNA and interacting proteins have remained elusive from a biochemical and structural perspective, mainly due to the technical difficulties in isolating and visualizing these transient, unstable, and highly dynamic particles, which exist in low quantities within the cell. The GOVERNA grant aims to develop biochemical strategies to isolate mRNPs for mechanistic studies and to obtain insight into their 3D organization by leveraging and integrating state-of-the-art approaches in structural biology.
In GOVERNA, our goals were to understand how mRNPs are constructed, how their composition and 3D organization evolve as they move through different cellular compartments and how they are recognized by cellular machineries. To address these goals and establish mechanistic principles that were applicable in general, we selected a broad range of mRNP targets that represented different levels of complexity or were present at different stages of the mRNA lifecycle. With these targets in mind, we then added modifications to help us purify the mRNPs in order to perform various biochemical assessments or subject the sample to cryogenic-electron microscopy (cryo-EM) or -electron tomography (cryo-ET).
We first sought to isolate mRNPs from the nucleus of budding yeast, S. cerevisiae, to understand how they are packaged. We then combined cross linking mass spectrometry data from our purified mRNPs with AlphaFold predictions in an interdependent approach to garner a realistic perspective of the interactions occurring between the mRNA and proteins. Cryo-ET and biochemical validation further supported a model whereby the mRNP is packaged by the Yra1 protein. Given the evolutionary conservation of Yra1, we would expect a similar mechanism in human mRNPs. In further work, we have shown that different protein factors are involved in different phases of the mRNPs journey to the cytoplasm. Indeed, it is expected that the mRNP composition will change over its lifetime, but we questioned what changes were involved when mRNPs were formed incorrectly. An occurrence that has been linked to certain pathologies in humans. The resulting proteomic data indicate nuclear and translational control factors are involved that show links to the translational quality control pathway known as nonsense mediated decay. In related work we showed the downstream binding interactions of Upf1, the sentinel protein factor in nonsense mediated decay, to explain the molecular basis of the mRNP formation that leads to removal of the RNA. We also derived a structural model that explained how the collision of translating ribosomes on problematic RNA can be resolved though the formation of a super-complex that extracts the RNA from the ribosomes and feed into a degradation complex. Finally, we have attained mRNPs from developing organisms, and we plan to exploit our structural and biochemical toolbox to ascertain valuable insights into the chemical make-up of these endogenous particles.
We first sought to isolate mRNPs from the nucleus of budding yeast, S. cerevisiae, to understand how they are packaged. We then combined cross linking mass spectrometry data from our purified mRNPs with AlphaFold predictions in an interdependent approach to garner a realistic perspective of the interactions occurring between the mRNA and proteins. Cryo-ET and biochemical validation further supported a model whereby the mRNP is packaged by the Yra1 protein. Given the evolutionary conservation of Yra1, we would expect a similar mechanism in human mRNPs. In further work, we have shown that different protein factors are involved in different phases of the mRNPs journey to the cytoplasm. Indeed, it is expected that the mRNP composition will change over its lifetime, but we questioned what changes were involved when mRNPs were formed incorrectly. An occurrence that has been linked to certain pathologies in humans. The resulting proteomic data indicate nuclear and translational control factors are involved that show links to the translational quality control pathway known as nonsense mediated decay. In related work we showed the downstream binding interactions of Upf1, the sentinel protein factor in nonsense mediated decay, to explain the molecular basis of the mRNP formation that leads to removal of the RNA. We also derived a structural model that explained how the collision of translating ribosomes on problematic RNA can be resolved though the formation of a super-complex that extracts the RNA from the ribosomes and feed into a degradation complex. Finally, we have attained mRNPs from developing organisms, and we plan to exploit our structural and biochemical toolbox to ascertain valuable insights into the chemical make-up of these endogenous particles.
The revelations unfolding from GOVERNA hold great value towards establishing a foundation for our understanding of mRNP biology. Many aspects of mRNA have been thoroughly investigated, for example, the chemical composition at its creation or the molecular machinery that choreograph its demise. Yet, our knowledge of the time in between those milestones has been shrouded in mystery due to shortcomings in technology that have failed to allow appropriate handling of this sensitive biological material. Our work has aimed to bring the events of this liminal stage of mRNA out of the darkness. We have established breakthrough methodologies to enable our work, which we have further powered with cutting edge technologies in the form of machine learning protein predictive software (AlphaFold). The synergy of these tools is allowing us to gain unprecedented views into mRNPs at the molecular level and to understand their biological impact through biochemical assays—enabling us to address imperative questions regarding how mRNA is protected throughout its lifetime. Our work is uncovering unexpected avenues in mRNA surveillance and in mechanisms to protect mRNA when the environment inside the cell is inhospitable. These are things that will springboard further research as our view of the overall system becomes funnelled from grasping overarching principles towards unravelling the finer details. Furthermore, our results shed light on physiological mechanisms put in place over thousands of years to maintain this biological system. If we can elucidate this biological design, which can be deemed successful as evidenced by its conservation across eukaryotic species, this basic knowledge can then be turned towards the benefit of clinical intervention.