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.
