Periodic Reporting for period 4 - EXORICO (Exosome and ribosome coupling)
Période du rapport: 2022-04-01 au 2022-09-30
Gene expression is one of the most fundamental processes inside the cell. The process of taking genetic information from DNA, temporarily holding it in the form of RNA, and finally translating it into actionable subunits as protein. This process has been finely tuned to eliminate mistakes and unessential parts, as failure to do so results in disease and in some cases death. Two major molecular machineries that interact and coordinate the control of gene expression are the exosome and the ribosome. These interactions constitute two essential quality control mechanisms that occur in different compartments of the cell. In the nucleus, the exosome has a constructive function in the maturation of pre-ribosomes, while its cytoplasmic variant is destructive – it controls the degradation of mRNA after its translation at the ribosome. Despite many landmark studies revealing structural composition and functional roles of these two assemblies and evidence that strongly supported their coordinated actions, the caveat remained that these machines had yet to be visualized together. The ERC grant EXORICO aimed to break through this barrier by using the latest in imaging technology, the cryo-electron microscope (cryo-EM), combined with the crystal structures of the individual building blocks to generate models of the large molecular machines detailed to the atomic level.
During the course of EXORICO, we showed for the first time how the exosome, a central RNA degradation machine, physically interacts with protein translation machinery, the ribosome.
In the nuclear interaction with the ribosome precursor, the exosome degrades a protruding part of the pre-ribosome and thereby induces both compositional and structural changes. This interaction is transient and could only be observed because we were able to stall the process. To this end, an exosome with a mutated active site was engineered which halts the chemical reaction on the pre-ribosome and thus “freezes” the transient interaction. The resulting structural analysis showed how the exosome remodels the ribosome’s large subunit during maturation. The complex mechanistic investigation was performed using yeast as a model system. In further work we demonstrated that a functional human nuclear exosome can be reconstituted from its 14 building blocks and that it is remarkably similar to the yeast version, demonstrating a high conservation of an essential molecular machine in evolution.
In the cytosol, the exosome interacts with the mature, translating ribosome to control mRNA fate. Our studies using the yeast system showed that ribosomes bound to the exosome via a cytoplasmic cofactor, the Ski complex. This work served as a lauching point for us to characterize biochemically reconstituted human cytoplasmic ribosomes with human SKI bound to mRNA at high resolution. Strikingly, we identified a complex regulatory mechanism that controls the channeling of mRNA into the exosome, a mechanism employed by lower eukaryotes as well. Subsequent work revealed this mechanism is also used by exosome-cofactor complexes in the nucleus.
In the nuclear interaction with the ribosome precursor, the exosome degrades a protruding part of the pre-ribosome and thereby induces both compositional and structural changes. This interaction is transient and could only be observed because we were able to stall the process. To this end, an exosome with a mutated active site was engineered which halts the chemical reaction on the pre-ribosome and thus “freezes” the transient interaction. The resulting structural analysis showed how the exosome remodels the ribosome’s large subunit during maturation. The complex mechanistic investigation was performed using yeast as a model system. In further work we demonstrated that a functional human nuclear exosome can be reconstituted from its 14 building blocks and that it is remarkably similar to the yeast version, demonstrating a high conservation of an essential molecular machine in evolution.
In the cytosol, the exosome interacts with the mature, translating ribosome to control mRNA fate. Our studies using the yeast system showed that ribosomes bound to the exosome via a cytoplasmic cofactor, the Ski complex. This work served as a lauching point for us to characterize biochemically reconstituted human cytoplasmic ribosomes with human SKI bound to mRNA at high resolution. Strikingly, we identified a complex regulatory mechanism that controls the channeling of mRNA into the exosome, a mechanism employed by lower eukaryotes as well. Subsequent work revealed this mechanism is also used by exosome-cofactor complexes in the nucleus.
The findings above demonstrate a huge step forward towards “watching” cellular events. They provide snapshots of large macromolecular assemblies while they are executing cellular processes. Their unprecedented resolution allows researchers to investigate the mechanisms of interaction of molecular machines with molecular detail. They have also offered a first glimpse into the intrinsic “gatekeeping” function used to control the indiscriminate exosome.